Inhibitor scaffold for the inhibition of the enzyme phosphoenolpyruvate carboxykinase

ABSTRACT

A PEPCK inhibitor can include identifying a molecule that has a size capable of fitting into and interacting with the PEPCK binding site and at least one of the following: (a) a first terminal substituent having co-planar atoms acting as metal ligands to the active site metal ion PEPCK; (b) at least one of an atom or substituent at positions 2 or 3 from the first terminal substituent includes a neutral carbon center or include an oxygen, sulfur, selenium, or other atom with similar physiochemical properties; (c) at least one of an atom or substituent at positions 2 or 3 from the first terminal substituent is devoid of an electropositive atom or substituents; or (d) a second terminal substituent opposite of the first terminal substituent, said second terminal substituent having an atom that is a hydrogen boding acceptor and/or is negatively charged.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation-in-part of PCT applicationPCT/US2009/030761, filed Jan. 12, 2009 which claims benefit of U.S.Patent Application Ser. No. 61/020,570, filed Jan. 11, 2008, whichapplications are incorporated herein by specific reference in theirentirety.

This invention was made with government support under Grant Numbers NSFDMB 85-20311 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

BACKGROUND

Phosphoenolpyruvate carboxykinase (“PEPCK”) is an enzyme used in thenatural process of gluconeogenesis. It converts oxaloacetate intophosphoenolpyruvate and carbon dioxide. Whereas most reactions ofgluconeogenesis can use the glycolysis enzymes in the oppositedirection, the pyruvate kinase enzyme is irreversible. Therefore, theenzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase areused to provide an alternate path for effectively reversing its actions.The enzyme, PEPCK, interacts with oxaloacetate (OAA) andphosphoenolpyruvate (PEP).

In humans there are two isoforms of PEPCK; a cytosolic form cPEPCK(SwissProt P35558) and a mitochondrial isoform mPEPCK (SwissProt Q16822)which have 63.4% sequence identity, which are products of differentgenes (Beale, E. G., Chrapkiewicz, N. B., Scoble, H. A., Metz, R. J.,Quick, D. P., Noble, R. L., Donelson, J. E., Biemann, K., and Granner,D. K. (1985) Rat hepatic cytosolic phosphoenolpyruvate carboxykinase(GTP). Structures of the protein, messenger RNA, and gene. J Biol Chem260, 10748-10760; Utter, M. F., and Kolenbrander, H. M. (1972) in TheEnzymes (Boyer, P. D., Ed.) pp 117-168, Academic Press, New York;Weldon, S. L., Rando, A., Matathias, A. S., Hod, Y., Kalonick, P. A.,Savon, S., Cook, J. S., and Hanson, R. W. (1990) Mitochondrialphosphoenolpyruvate carboxykinase from the chicken. Comparison of thecDNA and protein sequences with the cytosolic isozyme. J Biol Chem 265,7308-7317). Both forms bind free divalent metal cations, in addition tothe metal-nucleotide complex. Although these isozymes share 63% identityand posses a virtually identical three-dimensional structure (Dunten,P., Belunis, C., Crowther, R., Hollfelder, K., Kammlott, U., Levin, W.,Michel, H., Ramsey, G. B., Swain, A., Weber, D., and Wertheimer, S. J.(2002) Crystal structure of human cytosolic phosphoenolpyruvatecarboxykinase reveals a new GTP-binding site. J Mol Biol 316, 257-264;Holyoak, T., Sullivan, S. M.; and Nowak, T. (2006) Structural Insightsinto the Mechanism of PEPCK Catalysis. Biochemistry 45, 8254-8263;Sullivan, S. M., and Holyoak, T. (2007) Structures of rat cytosolicPEPCK: Insight into the mechanism of phosphorylation and decarboxylationof oxaloacetic acid. Biochemistry 46, 10078-10088), they areimmunologically distinct. It is almost certainly the cytosolic formwhich is important in gluconeogenesis, as there is no known transportmechanism to move PEP from the mitochondrion to the cytosol, where it isneeded for gluconeogenesis.

As PEPCK acts at the junction between glycolysis and the Krebs cycle, itcauses decarboxylation of a C4 molecule, creating a C3 molecule. As thefirst committed step in gluconeogenesis, PEPCK decarboxylates andphosphorylates OAA for its conversion to PEP, when GTP is present. As aphosphate is transferred, the reaction results in a GDP molecule.

PEPCK is enhanced, both in terms of its production and activation, bymany factors. Transcription of the PEPCK gene is stimulated by glucagon,glucocorticoids, retinoic acid, and adenosine 3′,5′-monophosphate(cAMP), while it is inhibited by insulin. Of these factors, insulin, ahormone that is deficient in the case of diabetes, is considereddominant, as it inhibits the transcription of many of the stimulatoryelements. PEPCK activity is also inhibited by hydrazine sulfate, and theinhibition therefore decreases the rate of gluconeogenesis. Also, theuse of siRNA to inhibit PEPCK in a diabetic mouse model showselimination of the hyperglycemia upon PEPCK inhibition. Therefore, itwould be advantageous to find novel inhibitors of PEPCK and/or aninhibitor scaffold for preparing analogs and derivatives thereof fornovel inhibitors of PEPCK.

BRIEF SUMMARY

In one embodiment, the present invention can include a compound forinhibiting phosphoenolpyruvate carboxykinase (PEPCK) in a subject. Sucha compound can include a molecular structure configured for interactingwith a biding site of PEPCK so as to inhibit PEPCK. The PEPCK inhibitorcan be characterized by having a size capable of fitting into andinteracting with the PEPCK binding site and at least one of thefollowing: (a) a first terminal substituent having co-planar atomsacting as metal ligands to the active site metal ion PEPCK; (b) at leastone of an atom or substituent at positions 2 or 3 from the firstterminal substituent includes a neutral carbon center or include anoxygen, sulfur, selenium, or other atom with similar physiochemicalproperties; (c) at least one of an atom or substituent at positions 2 or3 from the first terminal substituent is devoid of an electropositiveatom or substituents; or (d) a second terminal substituent opposite ofthe first terminal substituent, said second terminal substituent havingan atom that is a hydrogen boding acceptor and/or is negatively charged.For example, the PEPCK inhibitor can include at least one, two, three,or four of (a), (b), (c), or (d).

In one embodiment, the first terminal substituent can be characterizedby any one or more of the following: at least one of the co-planar atomsof the first terminal substituent interacts with S286; the firstterminal substituent has at least two cis-planar groups; the cis-planargroups are independently selected from carbonyls, amines, sulfhydryls,alcohols, or combinations thereof; the oxygen, nitrogen, or sulfur atomsof the cis-planar groups have an oxidation state; or at least one of thecis-planar groups is a carboxyl group that interacts with S286.

In one embodiment, at least one of the atoms or substituents atpositions 2 or 3 from the first terminal substituent can becharacterized by any one or more of the following: is electron rich atomor substituent so as to interact with at least one of R87 or R405 (e.g.,shown in FIG. 4 for CMMP described below); is devoid of anelectropositive atom or substituent that inhibits an interaction withone of R87 or R405; includes a carbonyl, carboxylate, ketone, orsulfonate moiety; interacts with R405 and/or R87 through anelectrostatic interaction and/or hydrogen bonding (e.g., CMMP has thisinteraction); at least one of positions 2 or 3 from the first terminalsubstituent includes a neutral carbon center; or both positions 2 and 3include a neutral carbon center.

In one embodiment, the second terminal substituent can be characterizedby any one or more of the following: includes a hydrogen bondingacceptor so as to interact with Y235 and/or N403; includes a negativecharge so as to interact with Y235 and/or N403; has an edge-on aromaticinteraction with Y235; includes a carbonyl group; or is devoid of apositive charge.

In one embodiment, the distance between the first terminal substituentand the second terminal substituent is 5 backbone atoms or less. Thiscan be any one of 5, 4, 3, or 2 atoms in order to fit within the PEPCKbinding site.

In one embodiment, the PEPCK inhibitor is selected from the groupconsisting of 3-[(carboxycarbonyl)oxy]-3-oxopropanoic acid,3-[(carboxycarbonyl)oxy]propanoic acid,3-[(carboxymethyl)sulfanyl]-2-oxopropanoic acid,(5,6-dioxo-1,4-dioxan-2-yl)acetic acid,(5,6-dioxo-5,6-dihydro-1,4-dioxin-2-yl)acetic acid,(2-hydroxy-5,6-dioxo-1,4-dioxan-2-yl)acetic acid, salts thereof, acidsthereof, derivatives thereof, or combinations thereof.

In one embodiment, the PEPCK inhibitor is or includes features of one ofFormulas A, B, C, or D, or salt thereof, acid thereof, derivativethereof or combinations thereof so as to interact with M2+ active metalsite of PEPCK through interactions (a) and (b) of the structures ofFormula A, Formula B, Formula C, and Formula D shown below.

In one embodiment, the PEPCK inhibitor is devoid of being characterizedby at least one of the following: a methyl or methylene center which isincapable of interacting with R405; a size incapable of fitting withinthe binding pocket framed by R87, K244, G237, F333, R405, N403 and/orY235; a steric conflict with F333; or a positively charged functionalgroup incompatible with a positively charged active site of PEPCK.

In one embodiment, the PEPCK inhibitor has a structure of Formula C,Formula E, or Formula G, or derivative or salt thereof.

In one embodiment, the present invention can include a pharmaceuticalcomposition for inhibiting phosphoenolpyruvate carboxykinase (PEPCK) ina subject. Such a composition can include a pharmaceutically acceptablecarrier; and a therapeutically effective amount of a PEPCK inhibitor asdescribed herein. For example, the PEPCK inhibitor can be present in atherapeutically effective amount for treating, inhibiting, and/orhyperglycemia in the subject. The subject can be a diabetic patient orsomeone susceptible to becoming a diabetic patient.

In one embodiment, the present invention can include a method fordesigning a molecule capable of functioning as a PEPCK inhibitor. Such amethod can include identifying a molecule having one or more features ofa PEPCK inhibitor as described herein. This can include combining one ormore features that are described to provide favorable interactions withPEPCK. Such a molecule can then be synthesized and tested for affinityand selectivity with PEPCK.

In one embodiment, the present invention can include a method forinhibiting phosphoenolpyruvate carboxykinase (PEPCK) in a subject. Sucha method can include administering to the subject a composition having aPEPCK inhibitor as described herein. The PEPCK inhibitor can beadministered in a therapeutically effective amount for treating,inhibiting, and/or hyperglycemia in the subject. The subject can be adiabetic patient.

These and other embodiments and features of the present invention willbecome more fully apparent from the following description and appendedclaims, or may be learned by the practice of the invention as set forthhereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

To further clarify the above and other advantages and features of thepresent invention, a more particular description of the invention willbe rendered by reference to specific embodiments thereof which areillustrated in the appended drawings. It is appreciated that thesedrawings depict only illustrated embodiments of the invention and aretherefore not to be considered limiting of its scope. The invention willbe described and explained with additional specificity and detailthrough the use of the accompanying drawings in which:

FIGS. 1A-1F schematically illustrate the modes of inhibitor binding torat cPEPCK. Shown are: FIG. 1A) PEPCK-Mn²⁺-oxalate, FIG. 1B)PEPCK-Mn²⁺-phosphonoformate, FIG. 1C) PEPCK-Mn²⁺-PGA, FIG. 1D)PEPCK-Mn²⁺-PGA, FIG. 1E) PEPCK-Mn²⁺-phosphonopropionate, and FIG. 1F)PEPCK-Mn²⁺-sulfoacetate complexes. The dashed lines indicate potentialhydrogen bonds and metal-water interactions. In addition, a potentialsalt bridge between R405 and the sulfate of sulfoacetate is shown inFIG. 1F. All distances indicated in FIGS. 1A-1F are in Ångstroms. TheF_(o)-F_(c) density rendered at FIG. 1A) 2.7 σ, FIG. 1B) 3.3 σ, FIG. 1C)2.4 σ, FIG. 1D) 2.4 σ, FIG. 1E) 3.1 σ, and FIG. 1F) 2.8 σ prior to theinclusion of the ligands into the model is shown as a blue mesh. TheF_(o)-F_(c) density shown in FIG. 1D is the residual F_(o)-F_(c) densityafter refinement of the PGA conformation shown in FIG. 1C.

FIGS. 2A-2B show the stereoview of the OAA/PEP binding site of the lidopen form of rat cPEPCK. The shading of the protein surface wasgenerated according to the calculated electrostatic surface potential.The areas range from (2) (shading 1; −0.5V) to (4) (shading 2; +0.5V).The electrostatic surface is rendered semi-transparent illustrating theresidues discussed in the text that are important for substraterecognition. These residues are labeled and rendered asball-and-stick-models. Bound oxalate and sulfoacetate are shown as stickmodels demonstrating the boundaries of the OAA/PEP binding site. Theactive site manganese ion is labeled and rendered as a pink sphere. Forclarity, the electrostatic surface was omitted for the active sitemanganese ion. FIGS. 2A and 2B are merely a rotation of 6 degrees withrespect to each other.

FIG. 3 is a schematic representation of an allosteric binding site for3-mercaptopicolinic acid (MPA) based upon crystallographic studies ofcytosolic PEPCK. The active site of PEPCK is indicated by the activesite manganese ion (sphere). The allosteric site is illustrated by thebound position of MPA framed by the P-loop domain and the 514-533 loop.W516 and C288 are involved in MPA binding. The F517 residue that changesconformation upon binding to the allosteric site is shown.

FIG. 4 is a schematic representation of a crystal structure of3-carboxymethylmercaptopicolinic acid(3-(carboxymethylsulfanyl)pyridine-2-carboxylic acid; CMMP) bound toPEPCK bridging both OAA/PEP subsites.

FIG. 5 includes a graph that illustrates the inhibition data for CMMPagainst PEPCK, where the data illustrates the inhibition of PEPCK byCMMP and demonstrate that it is competitive in its binding with PEP. Theglobal fit of the data to a competitive inhibition model gives a Kivalue of 12 uM. The ‘I’ values given in the legend are theconcentrations of CMMP used in uM.

DETAILED DESCRIPTION

Studies to delineate the topography of the active sites of the isozymesof PEPCK have been performed using analogues of PEP or OAA, either asreversible inhibitors or as alternative substrates (Ash, D. E., Emig, F.A., Chowdhury, S. A., Satoh, Y., and Schramm, V. L. (1990) Mammalian andAvian Liver Phosphoenolpyruvate Carboxykinase—Alternate Subs; Guidinger,P. F., and Nowak, T. (1990) Analogs of oxalacetate as potentialsubstrates for phosphoenolpyruvate carboxykinase. Arch Biochem Biophys278, 131-141). Alkyl or halo derivatives of PEP at the third carbon,using the numbering system of Stubbe and Kenyon (Stubbe, J. A., andKenyon, G. L. (1972) Analogs of phosphoenolpyruvate. Substratespecificities of enolase and pyruvate kinase from rabbit muscle.Biochemistry 11, 338-345) have demonstrated stereospecificity of thereaction catalyzed by mPEPCK from avian liver (Duffy, T. H., and Nowak,T. (1985) 1H and 31P relaxation rate studies of the interaction ofphosphoenolpyruvate and its analogues with avian phosphoenolpyruvatecarboxykinase. Biochemistry 24, 1152-1160). Also, α-hydroxyl andα-sulfhydryl carboxylic acids have been found to be poor substrates forPEPCK phosphoryl transfer reaction (Ash et al. 1990).

The present invention has provided systematic substrate analogues of PEPand OAA that are reversible inhibitors of PEPCK, which inhibition wasassayed against PEP. With the exception of three compounds, the PEPCKinhibitors were bifunctional, being predominantly bicarboxylic acids,biphosphonic acids, or bisulfonic acids. Some of the bifunctionalcompounds are also phosphoryl or sulfonyl monocarboxylic acids. None isan amide, ester, or acyl halide because earlier studies have shown thatsuch analogues are usually poor alternative substrates or inhibitors(Stubbe at al. 1972).

Although some of the analogues screened and described herein have beenused previously to study the PEP/OAA binding site of PEP-dependentenzymes (Fitch, C. D., Chevli, R., and Jellinek, M. (1979)Phosphocreatine does not inhibit rabbit muscle phosphofructokinase orpyruvate kinase. J Biol Chem 254, 11357-11359; Izui, K., Matsuda, Y.,Kameshita, I., Katsuki, H., and Woods, A. E. (1983) Phosphoenolpyruvatecarboxylase of Escherichia coli. Inhibition by various analogs andhomologs of phosphoenolpyruvate. J Biochem (Tokyo) 94, 1789-1795; Janc,J. W., Cleland, W. W., and O'Leary, M. H. (1992) Mechanistic studies ofphosphoenolpyruvate carboxylase from Zea mays utilizing formate as analternate substrate for bicarbonate. Biochemistry 31, 6441-6446; and, J.W., O'Leary, M. H., and Cleland, W. W. (1992) A kinetic investigation ofphosphoenolpyruvate carboxylase from Zea mays. Biochemistry 31,6421-6426; Meyer, C. R., Rustin, P., and Wedding, R. T. (1989) A kineticstudy of the effects of phosphate and organic phosphates on the activityof phosphoenolpyruvate carboxylase from Crassula argentea. Arch BiochemBiophys 271, 84-97), such as mPEPCK, few have been evaluated assubstrate analogues of rat liver cPEPCK and none have been structurallycharacterized in complex with the GTP-dependent PEPCK isozyme. Many ofthe compounds, such as sulfoacetate, 2,2-dimethylsulfoaceate,methanediphosphonate, and 1,2-ethanediphosphonate, have not beenpreviously evaluated as analogues of OAA or PEP or as inhibitors ofPEPCK.

Some of the small molecule compounds utilized in the studies have beenshown to inhibit PEPCK, however, they also inhibit a number of differentenzymes as well (e.g., very little to no selectivity). For thosecompounds that have been demonstrated previously to inhibit PEPCK, theirmechanism of interaction with PEPCK was previously unknown. Therefore,rationale design of new compounds based upon the interactions of thesenon-selective compounds was impossible prior to the structural andkinetic studies described herein. Also, the notion that there are twosubsites within the same active site of PEPCK, one for PEP and one forOAA was unknown prior to the present studies. The data demonstrate thatsome of the molecules studied bind specifically to one or the other ofthe two possible subsites of PEPCK. This key observation allows new andunique compounds to be designed and synthesized so as to incorporatefeatures of recognition of PEPCK suggested by the characterization. APEPCK inhibitor can include one or more features of the individualcompounds into one molecule.

Structural-function analysis of PEPCK illustrates that the ability ofcompounds having certain features to inhibit PEPCK, where such featurescan be dependent upon the overall size, orientation, electronicproperties, and charge of the functional groups on the compounds.Structural characterizations of the compounds bound to PEPCK demonstratethat in general there are two distinct classes of competitive inhibitorsfor PEPCK. The first class includes compounds that mimic PEP binding tothe enzyme in an outer-sphere geometry with respect to the active sitemetal, and have an order of magnitude lower affinity for PEPCK than thesecond class of compounds that mimic the binding of OAA, which has beenfound to directly coordinate to the active site metal ion. The novelstructure-function analysis presented herein illustrates the mechanismof molecular recognition used by PEPCK for selective interaction, whichcan be exploited to develop novel and selective inhibitors of thisimportant enzyme. Thus, the information obtained from thestructural-function analysis can be used to design molecules that areselective inhibitors of PEPCK, and such selective molecules can be usedfor inhibiting PEPCK for therapeutic purposes.

The structure-function analysis identified novel mechanisms of molecularrecognition of PEP and OAA by cPEPCK. As such, the systematic evaluationof a variety of PEP and OAA analogues determined the features ofmolecules that can function as potential reversible inhibitors of theenzyme against PEP. The molecules that inhibit PEPCK in a competitivefashion were found to fall into two general classes. Those moleculesthat can be competitive inhibitors of PEPCK that mimic the bindinggeometry of PEP, namely phosphoglycolate and 3-phosphonopropionate, arefound to bind weakly (millimolar K_(i) values). In contrast, thosemolecules that can be competitive inhibitors that mimic the bindinggeometry of OAA (oxalate and phosphonoformate) coordinate directly tothe active site manganese ion, and bind an order of magnitude moretightly (micromolar K_(i) values). Additionally, competitive inhibitorsulfoacetate is found to be an outlier of these two classes, and bindsin a hybrid fashion utilizing modes of recognition of both PEP and OAAin order to achieve a micromolar inhibition constant in the absence ofdirect coordination to the active site metal. The kinetic studies incombination with the structural characterization of the fiveaforementioned competitive inhibitors demonstrates the molecularrequirements for high affinity binding of molecules to the active siteof PEPCK. Examples of these features include (1) cis-planar carbonylgroups for coordination to the active site metal, (2) a bridgingelectron rich atom at the position corresponding to the C2 methylenegroup of OAA to facilitate interactions with R405, (3) a carboxylate orsulfonate moiety at a position corresponding to the C1 carboxylate ofOAA, and (4) an edge-on aromatic interaction between a carboxylate andY235. Molecules can have one or more of the foregoing features in orderto effectively inhibit PEPCK. Additional features of a PEPCK inhibitorare described below.

Each inhibitor of PEPCK is a new compound that can be included in a newcomposition of matter. The described inhibitors specifically andpotently inhibit the enzyme PEPCK. The inhibitor inhibits the enzymethat is the rate limiting step in glucose synthesis. This will eliminatethe production of glucose by the liver, and thereby eliminate thehyperglycemic condition in the peripheral tissues that leads to all theknown diabetic complications including circulatory problems, includingheart disease, and blindness. Thus, the PEPCK inhibitor can be used to:inhibit a hyperglycemic condition in the peripheral tissues; inhibitdiabetic complications such as circulatory problems, including heartdisease, and blindness; and thereby treat, inhibit, and/or preventcomplications associated with diabetes.

In one embodiment, the inhibitor is designed to specifically inhibit theenzyme PEPCK over other enzymes, whose role in the metabolic productionof glucose is well characterized. The PEPCK activity increases underconditions of hyperglycemia in diabetic patients, and thus inhibition ofPEPCK can treat and/or inhibit hyperglycemia.

In one embodiment, the present invention can include a compound forinhibiting phosphoenolpyruvate carboxykinase (PEPCK) in a subject. Sucha compound can include a molecular structure configured for interactingwith a biding site of PEPCK so as to inhibit PEPCK.

The molecule can inhibit PEPCK so as to inhibit gluconeogenesis, whichin many instances is the rate-limiting step. Gluconeogenesis is a majorcontributor to hyperglycemia in diabetics, not just impaired peripheralglucose uptake, and PEPCK is greatly elevated in diabetes. Accordingly,lowering PEPCK activity through inhibitors that interact with andinhibit the function of PEPCK can lead to lowered blood glucose. Loweredblood glucose is beneficial to treat and/or inhibit hyperglycemia.

The molecule for inhibiting PEPCK (i.e., PEPCK inhibitor) can beselected to include the features for interacting with PEPCK. As such,various PEPCK inhibitors fall under the present invention. Additionally,the present invention can include a molecule that acts as a scaffold toallow for chemical substitutions that provide functionalities toparticipate in as many interactions with PEPCK as possible. Thus, thePEPCK inhibitor can have various geometric and physicochemicalproperties as described herein.

Also, the PEPCK inhibitor can include additional groups condensed ontothe structures described herein in order to minimize the charges andencourage membrane permeability.

Examples of chemical structure-function properties that may increase ordecrease the PEPCK binding inhibitors are shown below in Table 1 withrespect to Compounds 1-35. Any of the molecules shown to have beneficialproperties can be prepared into analogues thereof so as to utilize theproperties in the design of a PEPCK inhibitor.

In one embodiment, a PEPCK inhibitor can be designed and/or synthesizedso as to have a favorable chemical structure that binds with PEPCK,wherein the favorable chemical structure is a structure identifiedherein from at least a portion of a molecule type selected from thegroup consisting of dicarboxylates, phosphonyl monocarboxylates,phosphoryl monocarboxylates, sulfonyl monocarboxylates, sulfinylmonocarboxylates, diphosphoryls, disulfonates, epoxy compounds, aromaticcompounds, salts thereof, acids thereof, or combinations thereof. Themolecules that fall under the above-referenced groups are shown in Table1.

In one embodiment, a PEPCK inhibitor can include a PEPCK-associatingportion of a molecule selected from the group consisting of maleate,fumarate, itaconate, sulfosuccinate, 3-phosphonopropionate,sulfoacetate, 2,2-dimethyl sulfoacetate, 3-sulfopropionate,methanediphosphonate, 1,2-ethanediphosphonate, methanedisulfonate, andthe like.

In one embodiment, a PEPCK inhibitor is devoid of a portion chemicalfeature that inhibits an interaction with PEPCK. A molecule that hassuch a feature that inhibits an interaction with PEPCK can be selectedfrom the group consisting of 1,2-cyclopentanedicarboxylate,2-amino-5-phosphonovalerate, serine phosphate, threonine phosphate,cysteic acid, cysteine sulfinic acid, 1,2-ethanedisulfonate,phosphomycin, phenylphosphate, and p-nitrophenylphosphate.

The PEPCK inhibitor can be characterized by having a size capable offitting into and interacting with the PEPCK binding site and at leastone of the following: (a) a first terminal substituent having co-planaratoms acting as metal ligands to the active site metal ion PEPCK; (b) atleast one of an atom or substituent at positions 2 or 3 from the firstterminal substituent includes a neutral carbon center or include anoxygen, sulfur, selenium, or other atom with similar physiochemicalproperties; (c) at least one of an atom or substituent at positions 2 or3 from the first terminal substituent is devoid of an electropositiveatom or substituents; or (d) a second terminal substituent opposite ofthe first terminal substituent, said second terminal substituent havingan atom that is a hydrogen boding acceptor and/or is negatively charged.For example, the PEPCK inhibitor can include at least one, two, three,or four of (a), (b), (c), or (d). The characterizations described in(a), (b), (c), or (d) can be made with respect to any one of Compounds36-41, Formulas A-D, or based on a scaffold thereof (Compounds 1-35 areshown in Table 1 below).

As shown, the first terminal substituent, as shown on the right side ofFormulas A-D include interactions a,b with the M2+ metal ligand ofPEPCK. The second terminal substituent is shown in the left side ofFormulas A-D.

According to Formulas A-D, the PEPCK inhibitors can have an overallgeometry and/or stereoelectronic properties as shown in Formulas A-D andCompounds 36-41. M2+ represents the divalent cation bound at the PEPCKactive site and the dashed lines (a,b) represent the metal coordinationof the indicated atoms in the inhibitor to that same metal ion. A PEPCKinhibitor can have co-planar atoms (a,b) acting as metal ligands to theactive site metal ion. The co-planar atoms could be oxygen, nitrogen,selenium or other similar atoms with similar physiochemical properties.The atom positions 2 and/or 3 as shown in the representative structurescontaining oxygen or sulfur can further include other electronegativeatoms with similar physiochemical properties, and may be of anyoxidation state. Also, atom positions 2 and/or 3 can contain neutralcarbon centers. For example, one or both atom positions 2 and/or 3 caninclude atoms or substituents that are electronegative. Alternatively,one or both atom positions 2 and/or 3 can be carbons. Also, the atompositions 2 and/or 3 should be devoid of atoms or substituents that areelectropositive. A PEPCK inhibitor may combine any features of the threeindependent molecules shown in Formulas A-C (e.g., as shown in FormulaD) as to facilitate as many favorable interactions between enzyme andinhibitor as possible to achieve optimal specificity and potency. Thecentral backbone of the molecule scaffold (e.g., as shown in FormulaA-D) can be limited to 5 atoms in length or equivalently as shown andconstrained by the structural data defining the active site of PEPCK.

In one embodiment, the PEPCK inhibitor can have a structure of Formula Cor derivative thereof as shown in Formula E. The compound of Formula Cis 3-carboxymethylmercaptopicolinic acid(3-(carboxymethylsulfanyl)pyridine-2-carboxylic acid; CMMP), which hasnow been identified as a PEPCK inhibitor as shown in the figures anddescribed in the examples below. Based on the data for CMMP includingFIGS. 4 and 5, it is conceived that derivatives of Formula C, which areshown in Formula E, may have similar PEPCK inhibitor functionality.Particularly, the crystallographic data in FIG. 4 provides evidence thatthe compounds of Formula E may have similar function to CMMP:

In Formula E: R1 can be a hydrogen, halogen, C1, F, CH₃, CH₃CH₂, orhigher or lower substituted or unsubstituted straight chain or branchedaliphatic (e.g., C1-C10), where R1 is not electropositive (+ve) and canbe electronegative (−ve), neutral, and/or an hydrogen bond donor; R2 canbe hydrogen, CH₃CH₂, or higher or lower substituted or unsubstitutedstraight chain or branched aliphatic (e.g., C1-C10), and may be the sameor different from R1; R3 can be a carboxylic acid, amide, Formula F,sulphate, phosphate, or other hydrogen bond donor or have a positivecharge; X can be C, O, or N, such that X is an hydrogen bond donor; andn can be 0, 1, 2, or 3. Formula G shows another derivative of CMMP withR3 being carboxylic acid. Also, the sulfur atom can have an increasedoxidation state other than the oxidation state shown. Alternatively, thesulfur group can be replaced by oxygen or nitrogen.

The PEPCK inhibitors of Formula C, E, and G include features thatsatisfy certain criteria for PEPCK inhibitor design as described herein.Accordingly, these PEPCK inhibitors can be characterized by having asize capable of fitting into and interacting with the PEPCK binding siteand have following: (a) a first terminal substituent having co-planaratoms acting as metal ligands to the active site metal ion PEPCK (e.g.,the nitrogen in the ring along with the carboxylic acid group linked tothe ring); (b) at least one of an atom or substituent at positions 2 or3 from the first terminal substituent includes a neutral carbon centeror include an oxygen, sulfur, selenium, or other atom with similarphysiochemical properties (e.g., the sulfur atom); (c) at least one ofan atom or substituent at positions 2 or 3 from the first terminalsubstituent is devoid of an electropositive atom or substituents (e.g.,sulfur atom or R1 and R1 substituents); or (d) a second terminalsubstituent opposite of the first terminal substituent, said secondterminal substituent having an atom that is a hydrogen boding acceptorand/or is negatively charged (e.g., carboxylic acid group adjacent to R1and R2).

In one embodiment, the first terminal substituent can be characterizedby any one or more of the following: at least one of the co-planar atomsof the first terminal substituent interacts with S286; the firstterminal substituent has at least two cis-planar groups; the cis-planargroups are independently selected from carbonyls, amines, sulfhydryls,alcohols, or combinations thereof; the oxygen, nitrogen, or sulfur atomsof the cis-planar groups have an oxidation state; or at least one of thecis-planar groups is a carboxyl group that interacts with S286.

In one embodiment, at least one of the atoms or substituents atpositions 2 or 3 from the first terminal substituent can becharacterized by any one or more of the following: is electron rich atomor substituent so as to interact with at least one of R87 or R405; isdevoid of an electropositive atom or substituent that inhibits aninteraction with one of R87 or R405; includes a carbonyl, carboxylate,ketone, or sulfonate moiety; interacts with R405 and/or R87 through anelectrostatic interaction and/or hydrogen bonding; at least one ofpositions 2 or 3 from the first terminal substituent includes a neutralcarbon center; or both positions 2 and 3 include a neutral carboncenter.

In one embodiment, the second terminal substituent can be characterizedby any one or more of the following: includes a hydrogen bondingacceptor so as to interact with Y235 and/or N403; includes a negativecharge so as to interact with Y235 and/or N403; has an edge-on aromaticinteraction with Y235; includes a carbonyl group; or is devoid of apositive charge.

In one embodiment, the distance between the first terminal substituentand the second terminal substituent is 5 backbone atoms or less. Thiscan be any one of 5, 4, 3, or 2 atoms in order to fit within the PEPCKbinding site.

For example, the PEPCK inhibitor can be characterized by at least one ofthe following: (1) the first terminal substituent includes at least twocis-planar groups capable of coordinating to an active site metal ofPEPCK, examples are carbonyl groups, amines, sulfhydryl, alcohol or thelike, and can have oxygen, nitrogen or sulfur atoms of some oxidationstate; (2) a bridging electron rich atom at a position corresponding toa C2 methylene group of oxaloacetate to facilitate interactions withR405; (3) a carboxylate, ketone, carbonyl, sulfonate moiety or the likeat a position corresponding to a C1 carboxylate of oxaloacetate; and (4)an edge-on aromatic interaction between the second terminal substituentand Y235, where the second terminal substituent is a carbonyl (e.g.,carboxylate).

In another example, the PEPCK inhibitor can be characterized by at leastone, two, three, or four of the following: (1) a terminal carbonyl(e.g., carboxylate) so as interact with S286; (2) a bridging oxygen orsimilar electron rich atom between a C1 carboxylate and C3 carbonyl ofoxaloacetate that provides tight binding through electrostatic and/orhydrogen bonding with R405; (3) a carboxylate or sulfonate at a positioncorresponding to a C1 carboxylate of oxaloacetate that interacts withR87 and R405; (4) an aromatic and hydrogen bonding interaction at aposition corresponding to a C1 carboxylate of phosphoenolpyruvate withY235 and/or N403.

In one embodiment, the inhibitor can be selected from or includefeatures of 3-[(carboxycarbonyl)oxy]-3-oxopropanoic acid,3-[(carboxycarbonyl)oxy]propanoic acid,3-[(carboxymethyl)sulfanyl]-2-oxopropanoic acid,(5,6-dioxo-1,4-dioxan-2-yl)acetic acid,(5,6-dioxo-5,6-dihydro-1,4-dioxin-2-yl)acetic acid,(2-hydroxy-5,6-dioxo-1,4-dioxan-2-yl)acetic acid, salts thereof, acidsthereof, or combinations thereof.

In one embodiment, the PEPCK inhibitor is devoid of being characterizedby at least one of the following: a methyl or methylene center which, isincapable of interacting with R405; a size incapable of fitting withinthe binding pocket framed by R87, K244, G237, F333, R405, N403 and/orY235; a steric conflict with F333; or a positively charged functionalgroup incompatible with a positively charged active site of PEPCK.

In any of the compounds described herein, an aliphatic or backbonecarbon can be substituted with O, N, S, or P. Any aliphatic carbon canhave a hydrogen replaced with a halogen, Cl, F, CH₃, CH₃CH₂, or higheror lower substituted or unsubstituted straight chain or branchedaliphatic (e.g., C1-C10), an adamantyl (e.g., 2-adamantyl or adamantanederivative), or cycle or heterocycle selected from phenyl, pyridine,pyrimidine, pyrazine, 1,3,5-triazine, 1,2,4-triazine, quinoline,isoquinoline, acridine, phenanthrolines, benzoquinolines,phenathridines, phenazines, quinoxalines, quinazolines, phthalazines,pteridines, cinnolines, pyrroles, imidazoles, 1,2,3-triazoles, 1,2,4,triazoles, tetrazoles, isoxazoles, 1,3-thiazoles, benzimidazoles,indoles, indazoles, benzothiazoles, phenols, naphthols, 2-furan,3-furan, 2-thiophene, 3-thiophene, 2-pyrrole, 3-pyrrole, 2-oxazole,4-oxazole, 5-oxazole, 2-thiazole, 4-thiazole, 5-thiazole, 2-imidazole,4-imidazole, 5-imidazole, 3-isoxazole, 4-isoxazole, 5-isoxazole,3-isothiazole, 4-isothiazole, 5-isothiazole, 4-(1,2,3) oxadiazole,5-(1,2,3) oxadiazole, 4-(1,2,3) triazole, 5-(1,2,3) triazole, or2-(1,3,4) thiadiazole.

As used herein, the term “analog”, “analogue,” or “derivative” is meantto refer to a chemical compound or molecule made from a parent compoundor molecule by one or more chemical reactions. As such, an analog can bea compound with a structure similar to that of Compounds 36-41, FormulasA-D, or based on a scaffold thereof.

In one embodiment, each hydrogen on Compounds 36-41, Formulas A-D can beindependently replaced with an alkyl, aliphatic, straight chainaliphatic, aliphatic having a chain hetero atom, branched aliphatic,substituted aliphatic, cyclic aliphatic, heterocyclic aliphatic havingone or more hetero atoms, aromatic, heteroaromatic, polyaromatic,polyamino acids, peptides, polypeptides, combinations thereof, halogens,halo-substituted aliphatics, and the like.

As used herein, the term “aliphatic” is meant to refer to a hydrocarbylmoiety, such as an alkyl group, that can be straight or branched,saturated or unsaturated, and/or substituted or unsubstituted, which hastwenty or less carbons in the backbone. An aliphatic group may comprisemoieties that are linear, branched, cyclic and/or heterocyclic, andcontain functional groups such as ethers, ketones, aldehydes,carboxylates, and the like. Exemplary aliphatic groups include but arenot limited to substituted and/or unsubstituted groups of methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,octadecyl, nonadecyl, eicosyl, alkyl groups of higher number of carbonsand the like, as well as 2-methylpropyl, 2-methyl-4-ethylbutyl,2,4-diethylpropyl, 3-propylbutyl, 2,8-dibutyldecyl, 6,6-dimethyloctyl,6-propyl-6-butyloctyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl,2-ethylhexyl, and the like. The terms aliphatic or alkyl alsoencompasses alkenyl groups, such as vinyl, allyl, aralkyl and alkynylgroups.

Substitutions within an aliphatic group can include any atom or groupthat can be tolerated in the aliphatic moiety, including but not limitedto halogens, sulfurs, thiols, thioethers, thioesters, amines (primary,secondary, or tertiary), amides, ethers, esters, alcohols, oxygen, andthe like. The aliphatic groups can by way of example also comprisemodifications such as azo groups, keto groups, aldehyde groups, carbonylgroups, carboxyl groups, nitro, nitroso or nitrile groups, heterocyclessuch as imidazole, hydrazino or hydroxylamino groups, isocyanate orcyanate groups, and sulfur containing groups such as sulfoxide, sulfone,sulfide, and disulfide. Additionally, the substitutions can be viasingle, double, or triple bonds, when relevant or possible.

Further, aliphatic groups may also contain hetero substitutions, whichare substitutions of carbon atoms, by hetero atoms such as, for example,nitrogen, oxygen, phosphorous, or sulfur. As such, a linker comprised ofa substituted aliphatic can have a backbone comprised of carbon,nitrogen, oxygen, sulfur, phosphorous, and/or the like. Heterocyclicsubstitutions refer to alkyl rings having one or more hetero atoms.Examples of heterocyclic moieties include but are not limited tomorpholino, imidazole, and pyrrolidino.

As used herein, the term “aromatic” is meant to refer to molecule is onein which electrons are free to cycle around circular or cyclicarrangements of atoms, which are alternately singly and doubly bonded toone another. More properly, these bonds may be seen as a hybrid of asingle bond and a double bond, each bond in the ring being identical toevery other. Examples of aromatic compounds that can be present includebenzene, benzyl, toluene, xylene, and the like. The aromatic compoundcan include hetero atoms so as to be a hetero aromatic such as pyridine,furan, tetrahydrofuran, and the like. Also, an aromatic can be apolycyclic aromatic such as naphthalene, anthracene, phenanthrene,polycyclic aromatic hydrocarbons, indole, quinoline, isoquinoline, andthe like.

As used herein, the term “amine” is meant to refer to moieties that canbe derived directly or indirectly from ammonia by replacing one, two, orthree hydrogen atoms by other groups, such as, for example, alkylgroups. Primary amines have the general structures RNH₂ and secondaryamines have the general structure R₂NH. The term amine includes, but isnot limited to methylamine, ethylamine, propylamine, isopropylamine,aniline, cyclohexylamine, benzylamine, polycyclic amines, heteroatomsubstituted aryl and alkylamines, dimethylamine, diethylamine,diisopropylamine, dibutylamine, methylpropylamine, methylhexylamine,methylcyclopropylamine, ethylcylohexylamine, methylbenzylamine,methycyclohexylmethylamine, butylcyclohexylamine, morpholine,thiomorpholine, pyrrolidine, piperidine, 2,6-dimethylpiperidine,piperazine, and heteroatom substituted alkyl or aryl secondary amines.

As used herein, the term “halo” means fluoro, chloro, bromo, or iodo,preferably fluoro and chloro.

The present invention is a pharmaceutical agent for the prevention ofhyperglycemia in diabetic patients. This will provide a unique agent toalleviate the diabetic complications thus extending life and improvingthe quality of life of the millions suffering from diabetes. Theinhibitor can be selected based on a structure-based design of a centralmolecular scaffold that is useful for the selective and potentinhibition of the gluconeogenic enzyme PEPCK. The inhibitor scaffold andderivatives thereof can be effective therapeutics against thehyperglycemic condition exhibited by diabetics that leads to all theirsecondary complications including heart disease and blindness. Thestructure-based design imparts the inhibitor with selectivity for thetarget enzyme PEPCK. Thus, the inhibitor will only inhibit PEPCKminimizing side effects due to inhibition of enzymes that utilizesimilar metabolites. The basic inhibitor scaffold describes a small fourcarbon molecule whose molecular synthesis is straight forward andeconomical.

Current therapies for diabetic hyperglycemia are ineffective and wroughtwith side effects stemming from the fact that the biological target ofthese agents is not known and thus the agents lack specificity forenzymes involved in the metabolic pathway of glucose synthesis. Theinhibitor of the present invention is designed as a potent inhibitor ofPEPCK, and since it is a structurally designed inhibitor it is specificto the architecture of the PEPCK active site thus combining potency withspecificity which should alleviate side effects due to non-specificinhibition of other biological processes. In one embodiment, theinhibitor does not interact or negatively impact other enzymes outsideof the pathway of glucose synthesis.

In one embodiment, the inhibitor can be administered to a diabeticsubject in a therapeutically effective amount to treat, inhibit, and/oralleviate the hyperglycemic condition in the diabetic subject. Currenttreatment of diabetic hyperglycemia involves the use of metformin,sulfonylurease and thiazolidinediones. These therapies exhibitsignificant side effects include weight gain, gastrointestinal problems,and liver toxicity. Thus, the inhibitor can be administered to adiabetic subject without or lesser effects of weight gain,gastrointestinal problems, and liver toxicity.

Compounds according to the present invention may be used inpharmaceutical compositions having biological/pharmacological activityfor the inhibition of PEPCK. These compositions comprise an effectiveamount of any one or more of the compounds disclosed herein, optionallyin combination with a pharmaceutically acceptable additive, carrier, orexcipient. Also, the compounds can be combined and/or prepared intopharmaceutically acceptable salts. The compounds may also beco-administered with other therapeutic agents, such as other compoundsthat inhibit PEPCK. The effective amount can be a therapeuticallyeffective amount of the compound sufficient for use in treating,inhibiting, and/or hyperglycemia in diabetic patients.

As used herein, the terms “an effective amount”, “therapeutic effectiveamount”, or “therapeutically effective amount” shall mean an amount orconcentration of a compound according to the present invention which iseffective within the context of its administration or use. Thus, theterm “effective amount” is used throughout the specification to describeconcentrations or amounts of compounds according to the presentinvention which may be used to reduce hyperglycemia in diabeticpatients.

As used herein, the term “pharmaceutically acceptable excipient” meansan excipient that is useful in preparing a pharmaceutical compositionthat is generally safe, non-toxic and neither biologically nor otherwiseundesirable, and includes an excipient that is acceptable for veterinaryuse as well as human pharmaceutical use. A “pharmaceutically acceptableexcipient” as used in the specification and claims includes both one andmore than one such excipient.

As used herein, the term “pharmaceutically acceptable acid additionsalts” refers to those salts which retain the biological effectivenessand properties of the free bases, which are not biologically orotherwise undesirable, and which are formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and thelike. Any of Compounds 1-41 or derivatives thereof can be prepared as apharmaceutically acceptable salt.

Groups which form pharmaceutically acceptable acid addition saltsinclude amines, hydrazines, amidines, guanidines, substitutedaryl/heteroaryl and substituted alkyl groups that carry at least anitrogen bearing substituent such as amino, uanidine, amidino, uanidineand the like.

The compounds of the present invention can be formulated into apharmaceutically acceptable formulation. Such a composition can beuseful to prevent, alleviate, eliminate, or delay hyperglycemia indiabetic patients.

In embodiments of the present invention, the pharmaceutical compositioncomprises an active component and inactive components. The activecomponents are compounds described herein and theirderivatives/analogues. The inactive components are selected from thegroup consisting of excipients, carriers, solvents, diluents,stabilizers, enhancers, additives, adhesives, and combinations thereof.

The amount of the compound in a formulation can vary within the fullrange employed by those skilled in the art. Typically, the formulationwill contain, on a weight percent basis, from about 0.01-99.99 weightpercent of the compounds of the present invention based on the totalformulation, with the balance being one or more suitable pharmaceuticalexcipients. Preferably, the compounds are present at a level of about1-80 weight percent.

Pharmaceutical preparations include sterile aqueous or non-aqueoussolutions, suspensions and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oil such as oliveoil, injectable organic esters such as ethyloliate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers, (such as those based onRinger's dextrose), and the like. Preservatives and other additives mayalso be present such as, for example, antimicrobials, antioxidants,chelating agents and inert gases and the like. Those of skill in the artcan readily determine the various parameters for preparing thesepharmaceutical compositions without resort to undue experimentation.

Pharmacological compositions may be prepared from water-insolublecompounds, or salts thereof, such as aqueous base emulsions. In suchembodiments, the pharmacological composition will typically contain asufficient amount of pharmaceutically acceptable emulsifying agent toemulsify the desired amount of the pharmacological agent. Usefulemulsifying agents include, but are not limited to, phosphatidylcholines, lecithin, and the like.

Additionally, the compositions may contain other additives, such aspH-adjusting additives. In particular, useful pH-adjusting agentsinclude acids, such as hydrochloric acid, bases or buffers, such assodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodiumborate, or sodium gluconate.

Furthermore, pharmacological agent compositions may, though not always,contain microbial preservatives. Microbial preservatives that may beemployed include, but are not limited to, methylparaben, propylparaben,and benzyl alcohol. The microbial preservative may be employed when thepharmacological agent formulation is placed in a vial designed formulti-dose use. Pharmacological agent compositions for use in practicingthe subject methods may be lyophilized using techniques well known inthe art.

The compositions may also include components, such as cyclodextrins, toenhance the solubility of one or more other components included in thecompositions. Cyclodextrins are widely known in the literature toincrease the solubility of poorly water-soluble pharmaceuticals or drugsand/or enhance pharmaceutical/drug stability and/or reduce unwanted sideeffects of pharmaceuticals/drugs. For example, steroids, which arehydrophobic, often exhibit an increase in water solubility of one orderof magnitude or more in the presence of cyclodextrins. Any suitablecyclodextrin component may be employed in accordance with the presentinvention. The useful cyclodextrin components include, but are notlimited to, those materials which are effective in increasing theapparent solubility, preferably water solubility, of poorly solubleactive components and/or enhance the stability of the active componentsand/or reduce unwanted side effects of the active components. Examplesof useful cyclodextrin components include, but are not limited to:β-cyclodextrin, derivatives of β-cyclodextrin,carboxymethyl-β-cyclodextrin, carboxymethyl-ethyl-β-cyclodextrin,diethyl-β-cyclodextrin, dimethyl-β-cyclodextrin, methyl-β-cyclodextrin,random methyl-β-cyclodextrin, glucosyl-β-cyclodextrin,maltosyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin,hydroxypropyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, and thelike and mixtures thereof.

The specific cyclodextrin component selected should have propertiesacceptable for the desired application. The cyclodextrin componentshould have or exhibit reduced toxicity, particularly if the compositionis to be exposed to sensitive body tissue, for example, eye tissue, etc.Very useful β-cyclodextrin components include β-cyclodextrin,derivatives of β-cyclodextrin and mixtures thereof. Particularly usefulcyclodextrin components include sulfobutylether β-cyclodextrin,hydroxypropyl cyclodextrin and mixtures thereof. Sulfobutyletherβ-cyclodextrin is especially useful, for example, because of itssubstantially reduced toxicity.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Examples ofsuitable excipients can include, but are not limited to, the following:acidulents, such as lactic acid, hydrochloric acid, and tartaric acid;solubilizing components, such as non-ionic, cationic, and anionicsurfactants; absorbents, such as bentonite, cellulose, and kaolin;alkalizing components, such as diethanolamine, potassium citrate, andsodium bicarbonate; anticaking components, such as calcium phosphatetribasic, magnesium trisilicate, and talc; antimicrobial components,such as benzoic acid, sorbic acid, benzyl alcohol, benzethoniumchloride, bronopol, alkyl parabens, cetrimide, phenol, phenylmercuricacetate, thimerosol, and phenoxyethanol; antioxidants, such as ascorbicacid, alpha tocopherol, propyl gallate, and sodium metabisulfite;binders, such as acacia, alginic acid, carboxymethyl cellulose,hydroxyethyl cellulose; dextrin, gelatin, guar gum, magnesium aluminumsilicate, maltodextrin, povidone, starch, vegetable oil, and zein;buffering components, such as sodium phosphate, malic acid, andpotassium citrate; chelating components, such as EDTA, malic acid, andmaltol; coating components, such as adjunct sugar, cetyl alcohol,polyvinyl alcohol, carnauba wax, lactose maltitol, titanium dioxide;controlled release vehicles, such as microcrystalline wax, white wax,and yellow wax; desiccants, such as calcium sulfate; detergents, such assodium lauryl sulfate; diluents, such as calcium phosphate, sorbitol,starch, talc, lactitol, polymethacrylates, sodium chloride, and glycerylpalmitostearate; disintegrants, such as colloidal silicon dioxide,croscarmellose sodium, magnesium aluminum silicate, potassiumpolacrilin, and sodium starch glycolate; dispersing components, such aspoloxamer 386, and polyoxyethylene fatty esters (polysorbates);emollients, such as cetearyl alcohol, lanolin, mineral oil, petrolatum,cholesterol, isopropyl myristate, and lecithin; emulsifying components,such as anionic emulsifying wax, monoethanolamine, and medium chaintriglycerides; flavoring components, such as ethyl maltol, ethylvanillin, fumaric acid, malic acid, maltol, and menthol; humectants,such as glycerin, propylene glycol, sorbitol, and triacetin; lubricants,such as calcium stearate, canola oil, glyceryl palmitostearate,magnesium oxide, poloxymer, sodium benzoate, stearic acid, and zincstearate; solvents, such as alcohols, benzyl phenylformate, vegetableoils, diethyl phthalate, ethyl oleate, glycerol, glycofurol, for indigocarmine, polyethylene glycol, for sunset yellow, for tartazine,triacetin; stabilizing components, such as cyclodextrins, albumin,xanthan gum; and tonicity components, such as glycerol, dextrose,potassium chloride, and sodium chloride; and mixture thereof. Excipientsinclude those that alter the rate of absorption, bioavailability, orother pharmacokinetic properties of pharmaceuticals, dietarysupplements, alternative medicines, or nutraceuticals.

Other examples of suitable excipients, binders and fillers are listed inRemington's Pharmaceutical Sciences, 18th Edition, ed. Alfonso Gennaro,Mack Publishing Co. Easton, Pa., 1995 and Handbook of PharmaceuticalExcipients, 3rd Edition, ed. Arthur H. Kibbe, American PharmaceuticalAssociation, Washington D.C. 2000, both of which are incorporated hereinby reference.

In some embodiments, the compounds in the compositions may be present asa pharmaceutically acceptable salt. The term “pharmaceuticallyacceptable salts” includes salts of the composition, prepared, forexample, with acids or bases, depending on the particular substituentsfound within the composition and the treatment modality desired.Pharmaceutically acceptable salts can be prepared as alkaline metalsalts, such as lithium, sodium, or potassium salts; or as alkaline earthsalts, such as beryllium, magnesium or calcium salts. Examples ofsuitable bases that may be used to form salts include ammonium, ormineral bases such as sodium hydroxide, lithium hydroxide, potassiumhydroxide, calcium hydroxide, magnesium hydroxide, and the like.Examples of suitable acids that may be used to form salts includeinorganic or mineral acids such as hydrochloric, hydrobromic,hydroiodic, hydrofluoric, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, phosphorous acids and the like. Other suitableacids include organic acids, for example, acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, glucuronic, galactunoric, salicylic, formic,naphthalene-2-sulfonic, and the like. Still other suitable acids includeamino acids such as arginate, aspartate, glutamate, and the like.

In general, pharmaceutically acceptable carriers for are well-known tothose of ordinary skill in the art. This carrier can be a solid orliquid and the type is generally chosen based on the type ofadministration being used. Suitable pharmaceutical carriers are, inparticular, fillers, such as sugars, for example lactose, sucrose,mannitol or sorbitol, cellulose preparations and/or calcium phosphates,for example tricalcium phosphate or calcium hydrogen phosphate,furthermore, binders such as starch paste, using, for example, corn,wheat, rice or potato starch, gelatin, tragacanth, methylcelluloseand/or polyvinylpyrrolidone, if desired, disintegrants, such as theabovementioned starches, furthermore carboxymethyl starch, crosslinkedpolyvinylpyrrolidone, agar, alginic acid or a salt thereof, such assodium alginate; auxiliaries are primarily glidants, flow-regulators andlubricants, for example silicic acid, talc, stearic acid or saltsthereof, such as magnesium or calcium stearate, and/or polyethyleneglycol. Sugar-coated tablet cores are provided with suitable coatingswhich, if desired, are resistant to gastric juice, using, inter alia,concentrated sugar solutions which, if desired, contain gum arabic,talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide,coating solutions in suitable organic solvents or solvent mixtures or,for the preparation of gastric juice-resistant coatings, solutions ofsuitable cellulose preparations, such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate. Colorants or pigments, forexample, to identify or to indicate different doses of activeingredient, may be added to the tablets or sugar-coated tablet coatings.

Additional pharmaceutically acceptable carriers that may be used inthese pharmaceutical compositions include, but are not limited to, ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as prolaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

Additional formulations for use in the present invention can be found inRemington's Pharmaceutical Sciences (Mack Publishing Company,Philadelphia, Pa., 17th ed. (1985)), which is incorporated herein byreference. Moreover, for a brief review of methods for drug delivery,see, Langer, Science 249:1527-1533 (1990), which is incorporated hereinby reference. The pharmaceutical compositions described herein can bemanufactured in a manner that is known to those of skill in the art,i.e., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes. Other examples of suitable pharmaceuticals arelisted in 2000 Med Ad News 19:56-60 and The Physicians Desk Reference,53rd edition, 792-796, Medical Economics Company (1999), both of whichare incorporated herein by reference.

In general, compounds of this invention can be administered aspharmaceutical compositions by any one of the following routes: oral,systemic (e.g., transdermal, intranasal or by suppository), orparenteral (e.g., intramuscular, intravenous or subcutaneous)administration. One manner of administration is oral using a convenientdaily dosage regimen which can be adjusted according to the degree ofaffliction. Compositions can take the form of tablets, pills, capsules,semisolids, powders, sustained release formulations, solutions,suspensions, elixirs, aerosols, or any other appropriate compositions.Another manner for administering compounds of this invention isinhalation.

Suitable preparations for parenteral administration are primarilyaqueous solutions of an active ingredient in water-soluble form, forexample a water-soluble salt, and furthermore suspensions of the activeingredient, such as appropriate oily injection suspensions, usingsuitable lipophilic solvents or vehicles, such as fatty oils, forexample sesame oil, or synthetic fatty acid esters, for example ethyloleate or triglycerides, or aqueous injection suspensions which containviscosity-increasing substances, for example sodiumcarboxymethylcellulose, sorbitol and/or dextran, and, if necessary, alsostabilizers.

Suitable rectally utilizable pharmaceutical preparations are, forexample, suppositories, which consist of a combination of the activeingredient with a suppository base. Suitable suppository bases are, forexample, natural or synthetic triglycerides, paraffin hydrocarbons,polyethylene glycols or higher alkanols. Furthermore, gelatin rectalcapsules which contain a combination of the active ingredient with abase substance may also be used. Suitable base substances are, forexample, liquid triglycerides, polyethylene glycols or paraffinhydrocarbons.

Recently, pharmaceutical formulations have been developed especially fordrugs that show poor bioavailability based upon the principle thatbioavailability can be increased by increasing the surface area i.e.,decreasing particle size. For example, U.S. Pat. No. 4,107,288 (hereinincorporated by reference) describes a pharmaceutical formulation havingparticles in the size range from 10 to 1,000 nm in which the activematerial is supported on a crosslinked matrix of macromolecules. U.S.Pat. No. 5,145,684 (herein incorporated by reference) describes theproduction of a pharmaceutical formulation in which the drug substanceis pulverized to nanoparticles (average particle size of 400 nm) in thepresence of a surface modifier and then dispersed in a liquid medium togive a pharmaceutical formulation that exhibits remarkably highbioavailability.

According to the methods of the present invention, the compositions ofthe invention can be administered by injection by gradual infusion overtime or by any other medically acceptable mode. Any medically acceptablemethod may be used to administer the composition to the patient. Theparticular mode selected will depend of course, upon factors such as theparticular drug selected, the severity of the state of the subject beingtreated, or the dosage required for therapeutic efficacy. The methods ofthis invention, generally speaking, may be practiced using any mode ofadministration that is medically acceptable, meaning any mode thatproduces effective levels of the active composition without causingclinically unacceptable adverse effects.

The administration may be localized (i.e., to a particular region,physiological system, tissue, organ, or cell type) or systemic. Forexample, the composition may be administered through parental injection,implantation, orally, vaginally, rectally, buccally, pulmonary,topically, nasally, transdermally, surgical administration, or any othermethod of administration where access to the target by the compositionis achieved. Examples of parental modalities that can be used with theinvention include intravenous, intradermal, subcutaneous, intracavity,intramuscular, intraperitoneal, epidural, or intrathecal. Examples ofimplantation modalities include any implantable or injectable drugdelivery system. Oral administration may be used for some treatmentsbecause of the convenience to the patient as well as the dosingschedule. Compositions suitable for oral administration may be presentedas discrete units such as capsules, pills, cachettes, tables, orlozenges, each containing a predetermined amount of the active compound.Other oral compositions include suspensions in aqueous or non-aqueousliquids such as syrup, an elixir, or an emulsion.

For injection, the compounds can be formulated into preparations bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives. Preferably, the compounds can be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

For oral administration, the compounds can be formulated readily bycombining with pharmaceutically acceptable carriers that are well knownin the art. Such carriers enable the compounds to be formulated astablets, pills, dragees, capsules; emulsions, lipophilic and hydrophilicsuspensions, liquids, gels, syrups, slurries, suspensions and the like,for oral ingestion by a patient to be treated. Pharmaceuticalpreparations for oral use can be obtained by mixing the compounds with asolid excipient, optionally grinding a resulting mixture, and processingthe mixture of granules, after adding suitable auxiliaries, if desired,to obtain tablets or dragee cores. Suitable excipients are, inparticular, fillers such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas, or from propellant-free, dry-powder inhalers. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampules orin multidose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulator agents such as suspending,stabilizing and/or dispersing agents.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compounds can be encapsulated in a vehicle such as liposomes thatfacilitates transfer of the bioactive molecules into the targetedtissue, as described, for example, in U.S. Pat. No. 5,879,713 to Roth etal. and Woodle, et al., U.S. Pat. No. 5,013,556, the contents of whichare hereby incorporated by reference. The compounds can be targeted byselecting an encapsulating medium of an appropriate size such that themedium delivers the molecules to a particular target. For example,encapsulating the compounds within microparticles, preferablybiocompatible and/or biodegradable microparticles, which are appropriatesized to infiltrate, but remain trapped within, the capillary beds andalveoli of the lungs can be used for targeted delivery to these regionsof the body following administration to a patient by infusion orinjection.

Microparticles can be fabricated from different polymers using a varietyof different methods known to those skilled in the art. The solventevaporation technique is described, for example, in E. Mathiowitz, etal., J. Scanning Microscopy, 4, 329 (1990); L. R. Beck, et al., Fertil.Steril., 31, 545 (1979); and S. Benita, et al., J. Pharm. Sci., 73, 1721(1984). The hot-melt microencapsulation technique is described by E.Mathiowitz, et al., Reactive Polymers, 6, 275 (1987). The spray dryingtechnique is also well known to those of skill in the art. Spray dryinginvolves dissolving a suitable polymer in an appropriate solvent. Aknown amount of the compound is suspended (insoluble drugs) orco-dissolved (soluble drugs) in the polymer solution. The solution orthe dispersion is then spray-dried. Microparticles ranging between 1-10microns are obtained with a morphology which depends on the type ofpolymer used.

Microparticles made of gel-type polymers, such as alginate, can beproduced through traditional ionic gelation techniques. The polymers arefirst dissolved in an aqueous solution, mixed with barium sulfate orsome bioactive agent, and then extruded through a microdroplet formingdevice, which in some instances employs a flow of nitrogen gas to breakoff the droplet. A slowly stirred (approximately 100-170 RPM) ionichardening bath is positioned below the extruding device to catch theforming microdroplets. The microparticles are left to incubate in thebath to allow sufficient time for gelation to occur. Microparticleparticle size is controlled by using various size extruders or varyingeither the nitrogen gas or polymer solution flow rates.

Particle size can be selected according to the method of delivery whichis to be used, typically size IV injection, and where appropriate,entrapment at the site where release is desired.

In one embodiment, the liposome or microparticle has a diameter which isselected to lodge in particular regions of the body. For example, amicroparticle selected to lodge in a capillary will typically have adiameter of between 10 and 100, more preferably between 10 and 25, andmost preferably, between 15 and 20 microns. Numerous methods are knownfor preparing liposomes and microparticles of any particular size range.Synthetic methods for forming gel microparticles, or for formingmicroparticles from molten materials, are known, and includepolymerization in emulsion, in sprayed drops, and in separated phases.For solid materials or preformed gels, known methods include wet or drymilling or grinding, pulverization, classification by air jet or sieve,and the like.

Embodiments may also include administration of at least onepharmacological agent using a pharmacological delivery device such as,but not limited to, pumps (implantable or external devices), epiduralinjectors, syringes or other injection apparatus, catheter and/orreservoir operatively associated with a catheter, injection etc. Forexample, in certain embodiments a delivery device employed to deliver atleast one pharmacological agent to a subject may be a pump, syringe,catheter or reservoir operably associated with a connecting device suchas a catheter, tubing, or the like. Containers suitable for delivery ofat least one pharmacological agent to a pharmacological agentadministration device include instruments of containment that may beused to deliver, place, attach, and/or insert at least onepharmacological agent into the delivery device for administration of thepharmacological agent to a subject and include, but are not limited to,vials, ampules, tubes, capsules, bottles, syringes and bags.

Sterile injectable forms of the compositions of this invention may beaqueous or a substantially aliphatic suspension. These suspensions maybe formulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant.

The pharmaceutical compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Pharmacological agents may be delivered transdermally, by a topicalroute, formulated as applicator sticks, solutions, suspensions,emulsions, gels, creams, ointments, pastes, jellies, paints, powders,and aerosols. For example, embodiments may include a pharmacologicalagent formulation in the form of a discrete patch or film or plaster orthe like adapted to remain in intimate contact with the epidermis of therecipient for a period of time. For example, such transdermal patchesmay include a base or matrix layer, e.g., polymeric layer, in which oneor more pharmacological agent(s) are retained. The base or matrix layermay be operatively associated with a support or backing. Pharmacologicalagent formulations suitable for transdermal administration may also bedelivered by iontophoresis and may take the form of an optionallybuffered aqueous solution of the pharmacological agent compound.Suitable formulations may include citrate or bis/tris buffer (pH 6) orethanol/water and contain a suitable amount of active ingredient.Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For other topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical compositions can be formulatedin a suitable lotion or cream containing the active components suspendedor dissolved in one or, more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith or without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

Depending on the mode of administration, the pharmaceutical compositionwill preferably comprise from 0.05 to 99% by weight, more preferablyfrom 0.1 to 70% by weight of the active ingredient, and, from 1 to99.95% by weight, more preferably from 30 to 99.9% by weight of apharmaceutically acceptable carrier, all percentages being based on thetotal composition.

The compositions of the present invention may be given in dosages,generally at the maximum amount while avoiding or minimizing anypotentially detrimental side effects. The compositions can beadministered in effective amounts, alone or in a cocktail with othercompounds, for example, other compounds that can be used to treat and/orprevent hyperglycemia in diabetic patients. An effective amount isgenerally an amount sufficient to inhibit hyperglycemia in diabeticpatients.

In one embodiment of the present invention, therapeutically effectiveamounts of compounds of the present invention may range fromapproximately 0.05 to 50 mg per kilogram body weight of the recipientper day; preferably about 0.01-25 mg/kg/day, more preferably from about0.5 to 10 mg/kg/day. Thus, for administration to a 70 kg person, thedosage range would most preferably be about 35-70 mg per day.

In another embodiment of the present invention, dosages may be estimatedbased on the results of experimental models, optionally in combinationwith the results of assays of the present invention. Generally, dailyoral doses of active compounds will be from about 0.01 mg/kg per day to2000 mg/kg per day. Oral doses in the range of 10 to 500 mg/kg, in oneor several administrations per day, may yield suitable results. In theevent that the response of a particular subject is insufficient at suchdoses, even higher doses (or effective higher doses by a different, morelocalized delivery route) may be employed to the extent that patienttolerance permits. Multiple doses per day are also contemplated in somecases to achieve appropriate systemic levels of the composition. Dosageamount and interval may be adjusted individually to provide plasmalevels of the active compound which are sufficient to maintaintherapeutic effect. Preferably, therapeutically effective serum levelswill be achieved by administering multiple doses each day. In cases oflocal administration or selective uptake, the effective localconcentration of the drug may not be related to plasma concentration.One having skill in the art will be able to optimize therapeuticallyeffective local dosages without undue experimentation.

Although the exact dosage will be vary dependent upon the percentcomposition of the dosage of compounds of the present invention, in mostcases some generalizations regarding the dosage can be made. The dailydosage regimen for an adult human patient may be, for example, an oraldose of between 0.1 mg and 2000 mg of each active ingredient, preferablybetween 1 mg and 500 mg, e.g. 5 to 200 mg. In other embodiments, anintravenous, subcutaneous, or intramuscular dose of each activeingredient of between 0.01 mg and 100 mg, preferably between 0.1 mg and60 mg, e.g. 1 to 40 mg is used. In cases of administration of apharmaceutically acceptable salt, dosages may be calculated as the freebase. In some embodiments, the composition is administered 1 to 4 timesper day. Alternatively the compositions of the invention may beadministered by continuous intravenous infusion, preferably at a dose ofeach active ingredient up to 1000 mg per day. As will be understood bythose of skill in the art, in certain situations it may be necessary toadminister the compounds disclosed herein in amounts that exceed, oreven far exceed, the above-stated dosage range in order to effectivelyand aggressively treat particularly aggressive diseases or infections.In some embodiments, the compounds will be administered for a period ofcontinuous therapy, for example for a week or more, or for months oryears.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain themodulating effects, or minimal effective concentration (MEC). The MECwill vary for each compound but can be estimated from in vitro and invivo data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. However, HPLCassays or bioassays can be used to determine plasma concentrations.

Use of a long-term release implant may be particularly suitable in somecases. “Long-term release,” as used herein, means that the implant isconstructed and arranged to deliver therapeutic levels of thecomposition for at least 30 or 45 days, and preferably at least 60 or 90days, or even longer in some cases. Long-term release implants are wellknown to those of ordinary skill in the art, and include some of therelease systems described above.

Any suitable dosage may be administered. The compound, the carrier, andthe amount will vary widely depending on body weight, the severity ofthe condition being treated and other factors that can be readilyevaluated by those of skill in the art. Generally a dosage of betweenabout 1 mg per kg of body weight and about 100 mg per kg of body weightis suitable.

In pharmaceutical dosage forms, agents may be administered alone or withan appropriate association, as well as in combination, with otherpharmaceutically active compounds. As used herein, “administered with”means that at least one pharmacological agent and at least one otheradjuvant (including one or more other pharmacological agents) areadministered at times sufficiently close that the results observed areindistinguishable from those achieved when one pharmacological agent andat least one other adjuvant (including one or more other pharmacologicalagents) are administered at the same point in time. The pharmacologicalagent and at least one other adjuvant may be administered simultaneously(i.e., concurrently) or sequentially. Simultaneous administration may becarried out by mixing at least one pharmacological agent and at leastone other adjuvant prior to administration, or by administering thepharmacological agent and at least one other adjuvant at the same pointin time. Such administration may be at different anatomic sites or usingdifferent routes of administration. The phrases “concurrentadministration,” “administration in combination,” “simultaneousadministration” or “administered simultaneously” may also be usedinterchangeably and mean that at least one pharmacological agent and atleast one other adjuvant are administered at the same point in time orimmediately following one another. In the latter case, the at least onepharmacological agent and at least one other adjuvant are administeredat times sufficiently close that the results produced are synergisticand/or are indistinguishable from those achieved when the at least onepharmacological agent and at least one other adjuvant are administeredat the same point in time. Alternatively, a pharmacological agent may beadministered separately from the administration of an adjuvant, whichmay result in a synergistic effect or a separate effect. The methods andexcipients described herein are merely exemplary and are in no waylimiting.

Moreover, toxicity and therapeutic efficacy of the compounds describedherein can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., by determining the LD₅₀, (thedose lethal to 50% of the population), the ED₅₀ (the dosetherapeutically effective in 50% of the population), and EC₅₀ (theexcitatory concentration effective in 50% of the population). The doseratio between toxic and therapeutic effect is the therapeutic index andcan be expressed as the ratio between LD₅₀ and ED₅₀. Compounds whichexhibit high therapeutic indices are candidates for further development.The data obtained from these cell culture assays and animal studies canbe used in formulating a dosage range that is not toxic for use inhuman. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See, e.g.,Fingl et al., 1975, In: “The Pharmacological Basis of Therapeutics”, Ch.1, p. 1). Additionally, the EC₅₀ can be important to measure.

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and mammalian species treated,the particular compounds employed, and the specific use for which thesecompounds are employed. The determination of effective dosage levels,that is the dosage levels necessary to achieve the desired result, canbe accomplished by one skilled in the art using routine pharmacologicalmethods. Typically, human clinical applications of products arecommenced at lower dosage levels, with dosage level being increaseduntil the desired effect is achieved. Alternatively, acceptable in vitrostudies can be used to establish useful doses and routes ofadministration of the compositions identified by the present methodsusing established pharmacological methods.

The exact formulation, route of administration and dosage for thepharmaceutical compositions of the present invention can be chosen bythe individual physician in view of the patient's condition. (See e.g.,et al. 1975, in “The Pharmacological Basis of Therapeutics”, which ishereby incorporated herein by reference in its entirety, with particularreference to Ch. 1, p. 1). Typically, the dose range of the compositionadministered to the patient can be from about 0.5 to 1000 mg/kg of thepatient's body weight. The dosage may be a single one or a series of twoor more given in the course of one or more days, as is needed by thepatient. In instances where human dosages for compounds have beenestablished for at least some condition, the present invention will usethose same dosages, or dosages that are between about 0.1% and 500%,more preferably between about 25% and 250% of the established humandosage. Where no human dosage is established, as will be the case fornewly-discovered pharmaceutical compounds, a suitable human dosage canbe inferred from EC₅₀, ED₅₀ or ID₅₀ values, or other appropriate valuesderived from in vitro or in vivo studies, as qualified by toxicitystudies and efficacy studies in animals.

It should be noted that the attending physician would know how to andwhen to terminate, interrupt, or adjust administration due to toxicityor organ dysfunctions. Conversely, the attending physician would alsoknow to adjust treatment to higher levels if the clinical response werenot adequate (precluding toxicity). The magnitude of an administrateddose in the management of the disorder of interest will vary with theseverity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

Compounds disclosed herein can be evaluated for efficacy and toxicityusing known methods. For example, the toxicology of a particularcompound, or of a subset of the compounds, sharing certain chemicalmoieties, may be established by determining in vitro toxicity towards acell line, such as a mammalian, and preferably human, cell line. Theresults of such studies are often predictive of toxicity in animals,such as mammals, or more specifically, humans. Alternatively, thetoxicity of particular compounds in an animal model, such as mice, rats,rabbits, or monkeys, may be determined using known methods. The efficacyof a particular compound may be established using several recognizedmethods, such as in vitro methods, animal models, or human clinicaltrials. Recognized in vitro models exist for nearly every class ofcondition, including but not limited to hyperglycemia in diabeticpatients, cardiovascular disease, and various immune dysfunction.Similarly, acceptable animal models may be used to establish efficacy ofchemicals to treat such conditions. When selecting a model to determineefficacy, the skilled artisan can be guided by the state of the art tochoose an appropriate model, dose, and route of administration, andregime. Of course, human clinical trials can also be used to determinethe efficacy of a compound in humans.

EXPERIMENTAL

Various materials were used in the experiments to study thestructure-function of molecules to inhibit PEPCK. The compounds used inthis study are shown in Table 1, which includes their name and chemicalformulae. Compounds 1-6,8,9,12, 17-27, and 29 were from AldrichChemicals; Compounds 14, 15, and 32 were from Fluka; Compounds 10, 11,13, 16, and 33-35 were from Sigma; Compounds 7 and 23 were from Kodak;Compound 28 was from Mallinckrodt; Compound 30 was from Alfa; andCompound 31 was from Pfaltz and Bauer. All compounds were of the highestcommercially available purity. The buffers TES and HEPES were fromResearch Organics. DTT, PEP, IDP, and TRIS were from Sigma. OAA and NADHwere from Boehringer Mannheim.

Various enzymes were used in the experiments to study thestructure-function of molecules to inhibit PEPCK. Malate dehydrogenase(1200 units/mg; 50% glycerol solution, v:v) was from BoerhingerMannheim. PEPCK utilized for the kinetic studies was purified tohomogeneity from rat liver cytosol following published procedures(Colombo, G., Carlson, G. M., and Lardy, H. A. (1978)Phosphoenolpyruvate carboxykinase (guanosine triphosphate) from ratliver cytosol. Separation of homogeneous forms of the enzyme with highand low activity by chromatography on agarose-hexane-guanosinetriphosphate. Biochemistry 17, 5321-5329; Lewis, C. T., Seyer, J. M.,and Carlson, G. M. (1989) Cysteine 288: an essential hyperreactive thiolof cytosolic phosphoenolpyruvate carboxykinase (GTP). J Biol Chem 264,27-33), with the following modifications. Frozen livers from 24-h fastedmale Wystar rats were purchased from Pel-Freeze. The final purificationof the enzyme by affinity chromatography on agarose-adipic acid-GTP(Sigma) was performed by eluting with a NaCl step gradient (100 mM to500 mM, in 100 mM increments) in 10 mM. TES (pH 7.2), 7.5% glycerol(v:v), 0.2 mM EDTA. This procedure afforded enzyme of >95% purity, basedon SDS-PAGE analysis. The enzyme preparation routinely had a specificactivity of 16-24 μM of OAA formed/min/mg protein at 25° C. Solutions ofenzyme were saturated with nitrogen and stored at 5° C. Under theseconditions, the enzyme was stable for 2-3 weeks. The concentration ofPEPCK was determined spectrophotometrically, using a molar extinctioncoefficient of 1.15×10⁵. The enzyme utilized for the crystallographicstudies was recombinantly expressed in and purified from E. coli cellsas previously described (Sullivan, S. M., and Holyoak, T. (2007)Structures of rat cytosolic PEPCK: Insight into the mechanism ofphosphorylation and decarboxylation of oxaloacetic acid. Biochemistry46, 10078-10088).

Molecules were studied in order to identify molecular features forinteracting with an inhibiting PEPCK. The inhibition of PEPCK by variouscompounds was evaluated using a continuous spectrophotometric assay inwhich the coupling enzyme, malate dehydrogenase, reduced OAA to malateconcomitant with the oxidation of NADH to NAD⁺. The decrease inabsorbance at 340 nm was monitored using a Beckman DU-70Spectrophotometer equipped with a temperature controller. The standard1-mL reaction mixture for the production of OAA contained 56 mMHEPES-KOH buffer (pH 7.0), 1 mM IDP (or alternatively, 0.1 mM GDP), 23 Umalate dehydrogenase (1 unit of malate dehydrogenase is defined as 1mole malate produced/min/mg protein), 0.25 mM NADH, 2.3 mM MnCl₂, and 48mM NaHCO₃. Under these conditions, PEP was the varied substrate. WhenIDP was the varied substrate for the determination of the pattern ofinhibition for pyrophosphate, the fixed PEP concentration was 2 mM. Eachmolecule tested for interaction with and/or inhibition of PEPCK wasincluded in a solution freshly prepared in 50 mM HEPES-KOH (pH 7.0), andthese stock solutions were diluted in the same buffer, with the pH beingmaintained. All assay components except PEPCK were preincubated in thecuvette for 3 min at 35° C., the standard assay temperature. Thistemperature was used because, during pilot experiments with theinhibitor phosphonoformate, greater inhibition was observed at 35° C.than at 20° C. Similarly, Nowak and Mildvan (Nowak, T., and Mildvan, A.S. (1970) Stereoselective interactions of phosphoenolpyruvate analogueswith phosphoenolpyruvate-utilizing enzymes. J Biol Chem 245, 6057-6064)demonstrated that inhibition of yeast enolase by the PEP analoguephosphoglycolate was also temperature-dependent. In our study, thereaction was initiated by addition of enzyme (10 μL of a 0.3 μM or 1.5μM solution diluted with 10 mM TES (pH 7.2), 0.2 mM DTT, 0.2 mM EDTA,and 7.5% glycerol (v:v). The specific activity of enzyme in theinhibition studies is expressed as μmoles OAA formed/min/mg protein.Assays to determine patterns of inhibition were performed in triplicate.Unless otherwise stated, titration experiments to estimate theconcentration of inhibitor that caused 50% inhibition of PEPCK activitywere performed in duplicate. At the highest concentrations used withPEPCK, none of the compounds evaluated in this study affected theactivity of the coupling enzyme, malate dehydrogenase.

The kinetic parameters for the inhibition studies were best fit to theequations for competitive inhibition (Eq. 1) and noncompetitiveinhibition (Eq. 2), using the computer program and nomenclature ofCleland (Cleland, W. W. (1979) Statistical analysis of enzyme kineticdata. Methods Enzymol 63, 103-138).

v=VA/[K(1+1/K _(is))+A]  (Eq. 1)

v=VA/[K(1/K _(is))+A(1+1/K _(ii))]  (Eq. 2)

The parameters are defined as follows: v is the initial velocity, V isthe maximal rate of product formation in the absence of inhibitor, A isthe concentration of the variable substrate, K is the apparent Michaelisconstant for the varied substrate, and K_(is) and K_(ii) are theinhibition constants.

For weakly inhibitory analogues for which a complete inhibition patternwas not determined, Dixon plots were used to determine K_(i) valuesSegal, I. H. (1976) in Biochemical Calculations pp 246-273, John Wiley &Sons, New York.

Crystals of PEPCK used for data collection were grown by thehanging-drop method at 25° C. by mixing 4 μL of protein [containing 10mg/mL PEPCK, 25 mM HEPES (pH 7.5) and 1 mM DTT] with 2 μL mother liquor[0.1 M HEPES (pH 7.4) and 16-24% PEG 3350 and 0.5 μL of 0.1 M MnCl₂].The crystals of the various inhibitor complexes were obtained andcryoprotected simultaneously by transferring the crystals to 20 μL dropscontaining 25% PEG 3350, 10% PEG 400, 0.1 M Hepes pH 7.5, 2 mM MnCl₂,and 10 mM of oxalate, PGA, phosphonoformate, phosphonopropionate, orsulfoacetate for 1 hour prior to cryocooling in liquid nitrogen.

Data on the cryocooled crystals at −180° C. were collected using aRU-H3R rotating Cu anode X-ray generator with Blue Confocal OsmicMirrors and a Rigaku Raxis IV++ detector. All data was integrated andscaled with HKL-2000(21). Data statistics are presented in Table 2.

The new structures of the rat cPEPCK were determined by molecularreplacement using MOLREP (Vagin, A., and Teplyakov, A. (1997) MOLREP: anautomated program for molecular replacement. J Appl Cryst 30, 1022-1025)in the CCP4 (Bailey, S. (1994) The Ccp4 Suite—Programs for ProteinCrystallography. Acta Cryst D50, 760-763) package and the previouslydetermined structure of rat cPEPCK (PDB 2QEW). This molecularreplacement solution was refined using Refmac5 followed by manual modeladjustment and rebuilding using COOT (Emsley, P., and Cowtan, K. (2004)Coot: model-building tools for molecular graphics. Acta Cryst D60,2126-2132). Ligand, metal, and water addition and validation were alsoperformed in COOT. Inspection of the F_(o)-F_(c) maps indicated that inthe PEPCK-Mn²⁺-PGA structure, two conformations of bound PGA werepresent in each of the two molecules in the ASU. The occupancy of thetwo conformations was manually adjusted (0.5) to minimize positive andnegative difference density peaks in the maps. The occupancy andB-factors for the ligands are given in Table 2.

A final round of TLS refinement was performed for all models in Refmac5.A total of 15 groups were utilized per chain in the PEPCK-Mn²⁺-oxalate,PEPCK-Mn²⁺-phosphonoformate, PEPCK-Mn²⁺-phosphonopropionate, andPEPCK-Mn²⁺-sulfoacetatate structures while 10 groups per chain wereutilized in the refinement of the PEPCK-Mn²⁺-PGA structure. The optimumTLS groups were determined by submission of the pdb files to the TLSMDserver (skuld.bmsc.washington.edu/˜tlsmd/index.html; Painter, J., andMerritt, E. A. (2005) A molecular viewer for the analysis of TLSrigid-body motion in macromolecules. Acta Cryst D61, 465-471). Instructures containing two molecules in the ASU, tight NCS restraintswere utilized during the initial rounds of refinement and were removedduring the final stages of refinement. All the models have excellentstereochemistry as determined by PROCHECK (Laskowski, R. A., Macarthur,M. W., Moss, D. S., and Thornton, J. M. (1993) Procheck—a Program toCheck the Stereochemical Quality of Protein Structures. J Appl Cryst 26,283-291). Final model statistics are presented in Table 2.

The initial preliminary screening of the compounds listed in Table 1involved comparison of their structural similarities to OAA and PEP. Oneof the criteria in choosing inhibitor compounds was that the inhibitorsof PEPCK possess functional groups similar to those of the OAA and PEPsubstrates. Thus, with the exception of three compounds (Compounds33-35), the molecules screened were bifunctional (e.g., bicarboxylates,biphosphonates, or bisulfonates) or else monocarboxylates withphosphonyl or sulfonyl groups as additional anionic moieties. A secondcriterion was size (e.g., molecular volume and length). Bifunctionalcompounds with conjugated ring systems or with more than six backbonecarbons were excluded. Many of the compounds evaluated have not beenutilized previously, and thus represent new reversible inhibitors of ratliver cPEPCK.

Inhibition was initially screened using a Dixon plot at a fixed PEPconcentration of 40 μM (½ K_(m)) and inhibitors from 1.5 to 6.0 mM.Unless otherwise stated, those molecules that caused less than 30-40%inhibition were not further studied (Table 1), although their inabilityto inhibit will be discussed. For the molecules that caused greaterinhibition, the pattern of inhibition was determined using PEP or IDP asthe varied substrate (Table 3).

Some of the molecules that inhibit PEPCK include oxaloacetate analogues.Oxalate (Table 1, Compound 1) is a competitive inhibitor of PEPCK with aK_(i) of 89 μM (Table 3). This value is similar to that of the K_(m) forPEP (82 μM) determined under the same conditions (data not shown). In aprevious study, oxalate was also found to be a competitive inhibitorwith respect to OAA of the rat liver cPEPCK, with a K, equivalent to theK_(m) for OAA (Ash, D. E., Emig, F. A., Chowdhury, S. A., Satoh, Y., andSchramm, V. L. (1990) Mammalian and Avian Liver PhosphoenolpyruvateCarboxykinase—Alternate Substrates and Inhibition by Analogs ofOxaloacetate. J Biol Chem 265, 7377-7384). Because of oxalate'sresemblance to the enolate of pyruvate, a postulated reactionintermediate generated during the conversion of PEP to pyruvate (Reed,G. H., and Morgan, S. D. (1974) Kinetic and magnetic resonance studiesof the interaction of oxalate with pyruvate kinase. Biochemistry 13,3537-3541), this analogue has been previously studied and found to be areversible inhibitor of PEP-dependent enzymes. Crystallographic studiesof Anaerobiospirillum succiniciproducens (Cotelesage, J. J. H., Prasad,L., Zeikus, J. G., Laivenieks, M., and Delbaere, L. T. J. (2005) Crystalstructure of Anaerobiospirillum succiniciproducens PEP carboxykinasereveals an important active site loop. Int J Biochem Cell Biol 37,1829), in addition to the structure of rat PEPCK in complex with oxalatepresented here, demonstrate that oxalate binds to the active sitemanganese in a bidentate fashion directly coordinating to the metalthrough the C1 and C2 carbonyl oxygens in an identical orientation tothat of the central skeleton of the substrate OAA (FIGS. 1A-1F). Thisconformation leaves one water molecule coordinated to the active sitemetal, which is subsequently displaced by the γ-phosphoryl oxygen ofGTP. Itaconate, the vinyl analogue of OAA, did not inhibit (Table 1,Compound 7), suggesting that the C3 keto oxygen present in the substrate(replaced by a methylene group in itaconic acid) is essential for theinteraction of the ligand with PEPCK through direct coordination withthe active site Mn²⁺ ion. Although the complete patterns of inhibitionwere not determined for the poor inhibitors succinate and maleate (Table1, Compounds 3 and 5), K_(i) values were obtained from Dixon plots,using two concentrations of PEP (Table 3). Succinate had a K_(i) valuegreater than 8 mM, whereas that for maleate was approximately 2 mM.Fumarate, the trans-isomer of maleate, did not inhibit PEPCK (Table 1,Compound 6). The lack of inhibition by 1,2-cyclopentanedicarboxylate(Compound 8) was likely due to the bulkiness of its cyclic moiety. Theseresults are in agreement with previous findings in which putative OAAanalogues were usually poor inhibitors, with K_(i) values above 6 mM(Hebda, C. A., and Nowak, T. (1982) The purification, characterization,and activation of phosphoenolpyruvate carboxykinase from chicken livermitochondria. J Biol Chem 257, 5503-5514; Hebda, C. A., and Nowak, T.(1982) Phosphoenolpyruvate carboxykinase. Mn2+ and Mn2+ substratecomplexes. J Biol Chem 257, 5515-5522).

Taken together with the structural data, the relatively poor inhibitionby the putative OAA analogues used in this and other studiesdemonstrates that the enzyme is relatively intolerant of changes in thebicarboxylate structure. The structure of oxalate, in combination withthe lack of inhibition by itaconate and the other OAA analoguesdemonstrates clearly the importance of the two planar cis-carbonylgroups. The structural data demonstrate that these sp²-hybridizedcenters are necessary for the cis-planar geometry (O—C—C—O torsion=−9.6°that is required to displace the previously bound water molecules in aperfect example of entropy-entropy compensation. Further, this planargeometry is the only geometry that allows for the conjugation of thecarbonyl groups and the ability to delocalize electrons through themetal center.

With the exception of oxalate, all of the OAA analogues tested, whileretaining a bicarboxylate electronic structure, lack this centralfeature and therefore would be deficient in forming the OAA-likeconformation that appears to be a central motif for tight binding ofligands directly to the active site manganese ion. Note the poor/lack ofinhibition by malonate, maleate and succinate (Tables 1 and 3). Thisconclusion is further supported by previous work in which lactate,malate, nitrolactate, glycerate, thioglycolate, μ-chlorolactate, andglycolate, all of which contain an sp³ hybridized center alpha to theterminal carboxylate, are either poor substrates or poor inhibitors ofmPEPCK. Additional support for this conclusion comes from theobservation that only one carboxylate interacts with the active sitemanganese ion in the mPEPCK-Mn²⁺-malonate-Mn²⁺GDP structure in ageometry vastly different than that of OAA/oxalate.

While the cis-sp² carbonyls are necessary for micromolar inhibition,they are not the only requirement. As has been demonstrated previously,pyruvate (and its β-mercapto-, fluoro-, nitro-, andhydroxy-derivatives), glyoxylate, α-ketobutyrate, α-ketoglutarate andacetopyruvate are poor or non-inhibitory compounds, despite containingthe cis sp² hybridized carbonyl centers. While acetopyruvate andα-ketoglutarate are likely excluded from the active site due to theirlarge size, the other compounds are isoelectronic with OAA and would bepredicted to bind more tightly than is observed based upon the aboveconclusion. The structural data again provide an explanation for thisdramatic reduction of 100-1000 fold in binding affinity (K_(i)oxalate=5-89 μM (K_(i) pyruvate=9 mM).

Comparing the tight binding analogues oxalate and phosphonoformate, itis observed that they both posses an oxygen anion that forms two shorthydrogen bonds in addition to an electrostatic interaction with R405(FIGS. 1A-1F). In contrast, all of the poor inhibitors mentioned abovethat contain the correct cis sp² carbonyl structure either lack thisoxygen (i.e. glyoxylate) or have a methyl or methylene center, which isincapable of taking advantage of the R405 interactions that appear to beworth between 3-4 kcal mol⁻¹ of binding energy. As OAA contains amethylene center alpha to the sp² carbonyl, it raises the question ofhow OAA achieves the reasonable K_(m) of ˜2-5 μM if the aforementionedinteraction is necessary for tight binding. The structure with OAAdemonstrates that compensation for the lack of interaction between R405and the C2 methylene group arises from interactions between R405 and/orR87 with the C1 carboxylate of OAA. The other inhibitors either do notposses a C1 carboxylate (e.g., pyruvate and its derivatives andα-ketobutyrate) or the group is not spatially located to correctlyinteract in a similar fashion (e.g., α-ketoglutarate). This results inthe observed poor inhibition by these molecules even in the presence ofthe cis carbonyl structure. This conclusion is further supported by theobservation that β-sulfopyruvate is a tight binding inhibitor of mPEPCK,suggesting that the β-sulfo group effectively mimics the C1 carboxylateof OAA and therefore binds to the enzyme with a K_(i) similar to theK_(m) of OAA (K_(i)=19−138 μM).

Phosphonoformate (Table 1, Compound 10) is a competitive inhibitor ofPEPCK with a K_(i) of 231 μM (Table 3). While initially chosen as amimic of PEP, the structural data clearly show the interaction ofphosphonoformate in a mode similar to that observed for OAA and oxalate(FIG. 1B). This appears to be a result of the O3 of the phosphono groupand the C1 carbonyl forming the same central cis-planar oxygen geometry(O—C—P—O torsion=−14.6°) as in oxalate and OAA. In addition, consistentwith the mechanism of recognition discussed above, the other carboxylateand phosphonate oxygens of phosphonoformate form similar electrostaticand hydrogen bonding interactions with R87 and R405 to those of oxalate,resulting in similar micromolar inhibition (Table 3). The slightlygreater distortion from planar found in the cis carbonyl centers ofphosphonoformate as compared to oxalate may explain the approximately2-fold greater K_(i) observed with phosphonoformate.

It has been found that phosphoenolpyruvate analogues can be PEPCKinhibitors. Phosphoglycolate (PGA, Table 1, Compound 13) is acompetitive inhibitor of PEPCK, with a K_(i) value of 1.04 mM (Table 3).Because of its marked resemblance to PEP in terms of molecular geometry,volume, and identical functional groups, PGA has been used previously asan alternative substrate or reversible inhibitor of PEP-dependentenzymes. Not surprisingly, the structural data show a binding mode ofPGA that is similar in the general orientation of the ligand to that ofPEP, with the phosphate coordinating to the active site manganese ionand the ligand extending away from the ion toward Y235 (FIGS. 1C-1D).Unlike the other inhibitor complexes, the PGA bound to PEPCK isstatically disordered and found in two different conformations in eachmolecule in the ASU. While one conformation is virtually identical tothe bound conformation of 3-phosphonopropionate (Compound 12), the otherconformation is unique to PGA and has the phosphate group situated in aposition similar to its location in the PEPCK-Mn²⁺-PEP complex (data notshown). As 3-phosphonopropionate is the phosphonate analogue of PGA andthe two compounds are structurally similar, this additional conformationand static disorder in PGA must be due to the presence of the bridgingphosphate oxygen that is absent in 3-phosphonopropionate. Thisconclusion is confirmed by the structural data demonstrating that thepresence of the bridging phosphate oxygen atom in PGA results in theformation of a hydrogen bond with either the NH1 or NH2 group of R405 inthe two bound conformations respectively. The conformation of boundinhibitor that is shared by PGA and 3-phosphonopropionate appears to bethe result of the loss of the unsaturated C3 methylene group that ispresent in PEP. This removes an apparent aromatic interaction betweenthe methylene group and F333 and allows the PGA and3-phosphonopropionate inhibitors to shift in the binding pocket in adirection toward F333. This displacement in the conformation of boundPGA and 3-phosphonopropionate shown in FIGS. 1C-1D results in anadditional difference between the bound conformation of the inhibitorsand that of PEP. While PEP is found to interact indirectly with theactive site metal ion through two coordinating water molecules, onephosphate/phosphono oxygen of PGA and 3-phosphonopropionate displacesone of the metal coordinated water molecules, resulting in thephosphate/phosphono group coordinating directly to the manganese center.This change in metal coordination of the bound PGA and3-phosphonopropionate inhibitors results in the loss of theY235-carboxylate aromatic interaction and the hydrogen bond between thecarboxylate and the side chain amide of N403 that, based upon thedifference in the K_(m) for PEP (82 μM) and the K_(i) for the PGA and3-phosphonopropionate (1-2 mM), appear to be responsible for at least1.4 kcal mol⁻¹ of binding energy.

Phosphonoacetate (Compound 11), a methylene homologue ofphosphonoformate is noninhibitory; however as mentioned above, the nextlarger methylene homologue of phosphonoacetate, 3-phosphonopropionate(Compound 12), is inhibitory, with a K_(i) value of 1.9 mM (Table 3).The similar inhibitory capability of 3-phosphonopropionate and PGA(Table 3) is consistent with the similar size and hybridization state ofthe bridging methylene group and oxygen atom of the two compounds. Also,as discussed above, the structure of PEPCK in complex with3-phosphonopropionate shows a virtually identical bound conformation tothat of PGA, again consistent with their similar millimolar inhibitionconstants (FIG. 1E). Unlike what is observed with PGA versus3-phosphonopropionate, the methylene analogue of PEP,2-(phosphonomethyl)acrylate, is noninhibitory (Table 1, Compound 15).This lack of inhibition is most likely the result of the inability ofthis longer inhibitor to fit within the PEP binding pocket.

2-D-Phosphoglycerate is also noninhibitory (Table 1, Compound 14). Thisanalogue is similar to phosphoglycolate, except for the replacement of aC2 hydrogen with the larger —CH₂OH group, whose introduction wouldinterfere with binding at the PEP site through a steric effect. Anotheranalogue with a bulky group at the C2 position,2-amino-3-phosphonopropionate (Compound 18), likewise fails to inhibit,even though 3-phosphonopropionate is moderately inhibitory. Both2-amino-3-phosphonopropionate and 3-phosphonopropionate also have theadded negative factor of a methylene bridge between C2 and thephosphoryl group. The bridging methylene group in itself hinders binding(compare Compound 15 with PEP); nevertheless, a negative effect of aminosubstitution at C2 can also be observed by comparing Compounds 25 and26. Because the C2 carbons in 2-D-phosphoglycerate and2-amino-3-phosphonopropionate are chiral, one might infer that the lackof inhibition could be reversed by using the L-isomer, at least in thecase of phosphoglycerate. Studies have shown that the D- and L-isomersof PEP analogues inhibit some PEP-dependent enzymes differently;however, in the experiment using the noninhibitory2-amino-3-phosphonopropionate, a racemic mixture was used. No inhibitionis observed with any dicarboxylates or phosphonyl monocarboxylates thatcontain an amino group (Table 1, Compounds 9, and 18-22), although allof these compounds are rather bulky (relatively large volumes andlengths). The analogue 6-phosphonogluconate (Compound 16) also does notinhibit, presumably due to its increased molecular volume and length.The lack of inhibition by these compounds illustrates the tightgeometric constraints placed upon the substitution at the C2 carbon ofPEP. The active site pocket for PEP is framed by R87, K244, G237, F333,R405, N403 and Y235. The structural data suggest that substitutions atC2 are limited, as these substitutions in the location of the C2 protonof PEP would sterically conflict with R87. Similarly, substitutions atthe site corresponding to the C3 methylene group of PEP would seem to belimited to the native methylene group because of steric conflict withF333.

It has been found that sulfonyl monocarboxylates can be PEPCKinhibitors. A priori, these compounds were not categorized as OAAanalogues due to the different hybridization states and geometry oftheir terminal groups. The carbon atom in a carboxylate anion issp²-hybridized, whereas the sulfur atom can be considered essentiallysp³-hybridized (Reed, A. E., and Schleyer, P. V. (1990) Chemical Bondingin Hypervalent Molecules—the Dominance of Ionic Bonding and NegativeHyperconjugation over D-Orbital Participation. J Am Chem Soc 112,1434-1445). The carboxylate group is planar with C_(2v) geometry, asopposed to the nonplanar C_(3v) geometry of the sulfonyl group (Kanyo,Z. F., and Christianson, D. W. (1991) Biological recognition ofphosphate and sulfate. J Biol Chem 266, 4264-4268). Although theelectronic charge for both groups is the same (−1), the sulfonyl groupis more electron dense. The sulfonyl monocarboxylates were notcategorized as PEP analogues for similar reasons: a slight change inhybridization state (the phosphorous atom in a phosphoryl group has sp³hybridization and C_(3v) geometry), electron density, and a decrease of1 in net electronic charge (Radzicka, A., and Wolfenden, R. (1991)Analogues of intermediates in the action of pig kidney prolidase.Biochemistry 30, 4160-4164). As mentioned above, β-sulfopyruvate waspreviously reported to be a potent reversible inhibitor of avian livermPEPCK, with a K_(i) value of 7 μM. The analogue was competitive withrespect to OAA, with which it is isoelectronic. We have placed sulfonylmonocarboxylates in a separate class of PEPCK inhibitors, and maypotentially be considered as either OAA or PEP analogues, because thesulfonyl group of these compounds does not perfectly mimic either thecarboxyl or phosphoryl functionalities, and thus could potentiallycompete with substrates for binding at either phosphoryl- orcarboxyl-binding sites. However, as demonstrated by the structure ofPEPCK in complex with sulfoacetate, the sulfo-group appears, in the caseof recognition by PEPCK, to be better analogue of the carboxylate groupthan the phosphate group.

Sulfoacetate (Compound 23) is a competitive inhibitor of PEPCK withrespect to PEP, with a K_(i) value of 83 μM (Table 3), which is similarto the K_(m) value for PEP determined under these conditions. Incontrast to the other micromolar inhibitors, sulfoacetate does not mimicthe binding of OAA by coordinating directly to the active site metal, aswould be predicted by the absence of cis-planar carbonyl groups. Thisinhibitor instead binds in a hybrid orientation, mimicking elements ofboth OAA and PEP recognition. The carboxylate of sulfoacetate binds inan identical orientation to the C1 carboxylate of PEP forming the sameedge on interaction with Y235 and a hydrogen bond between thecarboxylate and N403. In contrast, the sulfo-group does not mimic thebinding of the phosphate of PEP; instead the sulfo-group is locatedsimilarly to that of the C1 carboxylate of OAA, interacting with R87 andR405 (FIG. 1F). Therefore, it is apparent that the combination of theedge on aromatic interaction and the hydrogen-bonding and electrostaticinteractions with R87/405 and N403 are sufficient to result in the tightbinding of the inhibitor in the absence of direct coordination to theactive site manganese ion.

The analogue of sulfoacetate, 2,2-dimethylsulfoacetate (Compound 24), isalso a competitive inhibitor, but with a K_(i) 25-fold greater than thatof sulfoacetate (Table 3). This decrease in binding affinity ispresumably due to the presence of the bulky methyl groups.3-Sulfonopropionate (Compound 25) is also a poor inhibitor (Table 3),but its amino analogue cysteic acid (Compound 26) is even lessinhibitory, whereas the related compound cysteine sulfinate (Compound27) does not cause inhibition. Sulfosuccinate (Compound 4) is a poorinhibitor, with a K_(i) value of approximately 3.3 mM (Table 3);however, this inhibition is noteworthy considering that the parentcompound succinate (Compound 3) is even less effective (Table 3).Sulfosuccinate and aspartate are both analogues of succinate, butsulfosuccinate and not aspartate (Table 1, Compound 9) inhibits; thisdifference is most likely due to the positive charge on aspartate and anegative charge on sulfosuccinate at the same carbon. These compoundsalso provide further evidence that the presence of an amino group on thecarbon atom a to the terminal carboxyl group inhibits binding. Nocompound tested in this study runs counter to this observation (Table1). These results are in agreement with a previous report that suggestedthat a change in the hybridization state of the C2 carbon of PEP fromsp² to sp³ or the incorporation of bulky groups at this positiondecreases affinity. Our structural data further support theseobservations, with the tight framing of the PEP binding pocket by F333and R87 allowing for relatively few changes at this site of the moleculeand the overall positive electrostatic potential at the active site ofPEPCK (FIGS. 2A-2B).

It has now been found that diphosphoryls and diphosphonates can be PEPCKinhibitors. Pyrophosphate (Compound 28) is a noncompetitive inhibitor ofPEPCK with respect to PEP, with a K_(is) value of 34 μM and a K_(ii)value of 63 μM. With IDP as the variable substrate, however, theinhibition pattern suggests that pyrophosphate is a competitiveinhibitor (K_(i) of 172 μM; Table 3), perhaps competing with the α- andβ-phosphates of IDP. The methylene analogue of pyrophosphate,methanediphosphonate (Compound 29), is also a noncompetitive inhibitorof PEPCK with respect to PEP, with inhibition constants similar to thoseof pyrophosphate (Table 3). This similarity in the inhibition patternsand constants observed for pyrophosphate and methanediphosphonatesuggests that both compounds may bind at the same site. Althoughmethanediphosphonate has not been previously reported as a reversibleinhibitor of PEPCK from any source, or as a reversible inhibitor of anyPEP-utilizing enzyme, it has been reported to be an inhibitor ofpyrophosphate-dependent phosphofructokinase, with an IC₅₀ of >2 mM. Thehomologue of methanediphosphonate, 1,2-ethanediphosphonate (Compound30), is a poor competitive inhibitor, with a K_(i), value of 5.1 mM(Table 3). The functional groups on 1,2-ethanediphosphonate are in thesame relative positions as those of phosphoglycolate,3-phosphonopropionate, and 3-sulfonopropionate, each of which is amoderate to poor inhibitor of the enzyme.

The corresponding sulfonate analogues of pyrophosphonate andmethanediphosphonate are pyrosulfate and methanedisulfonate (Compound31). Pyrosulfate cannot be evaluated as an inhibitor because it isinstantaneously converted to sulfuric acid in an aqueous environment;however, methanedisulfonate is stable and was found, like itsdiphosphonate analogue, to be a noncompetitive inhibitor (Table 3). Theinhibition of PEPCK by pyrophosphate was anticipated, based either onits ability to chelate divalent metal cation at the active site of themetal-dependent protein or on its mimicry of the oligophosphate sidechain of the nucleotide substrate (Harris, W. R., and Nesset-Tollefson,D. (1991) Binding of phosphonate chelating agents and pyrophosphate toapotransferrin. Biochemistry 30, 6930-6936). Thus, it can be predictedthat methanediphosphonate and methanedisulfonate, beinghomo-bifunctional compounds of similar size and configuration aspyrophosphate, may also be non-competitive inhibitors (Table 3,Compounds 29 and 31). Given that the gamma-phosphate of GTP acts abridging ligand between the active site and nucleotide metals, thesebi-functional compounds could be blocking the gamma phosphate'sinteraction with the active site metal, as this site has beencrystallographically observed to bind anions such as sulfate (Holyoakand Sullivan, unpublished data). It is therefore possible that theseshort bi-functional compounds are bridging the two metals by binding inthe γ- and β-phosphate binding site, thereby inhibiting phosphoryltransfer. The competitive inhibition of pyrophosphate against IDP isconsistent with this conclusion, as the pyrophosphate and IDP arecompeting at least partially for the same site.

Using the double inhibition approach of Janc et al., it was determinedwhether methanedisulfonate (noncompetitive inhibitor) and sulfoacetate(competitive inhibitor) could bind simultaneously to PEPCK. The paralleldouble inhibition patterns obtained with these two inhibitors (data notshown) are consistent with their binding being mutually exclusive.1,2-Ethanedisulfonate (Compound 32), a homologue of methanedisulfonate,also inhibits, but weakly, with a K_(i) value of approximately 3.0 mM(Table 3). The sulfonyl groups on 1,2-ethanedisulfonate are in the samerelative positions as the functional groups of the previously mentionedanalogues succinate, sulfosuccinate and 1,2-ethanediphosphonate. Becauseall of these compounds have similar structures and are poor-to-moderateinhibitors, the decrease in affinity most likely results from theirinability to mimic the bound conformation of OAA, based upon thequalities previously discussed. They are, instead, predicted to bind ina fashion similar to PEP, PGA and 3-phosphonopropionate.

In general, those molecules that fail to inhibit PEPCK are predicted tobe sterically prohibited from binding to the active site and/or carrypositively charged functional groups incompatible with the positivelycharged active site (FIGS. 2A-2B). Of the molecules that demonstratesome affinity for the enzyme (Table 3), the structural data suggest thatthe phosphoryl- and phosphono-monocarboxylates attain the correctpolarity in the active site via the phosphoryl/phosphono group,orienting the phosphorus containing moiety toward the manganese centerand positioning the carboxylate towards the end of the pocket and Y235and N403. These are weak inhibitors that in a general sense mimic thebinding orientation of PEP; however, they appear to have a reducededge-on carboxylate interaction with Y235 and lose the hydrogen bondinginteraction with N403 as the result of movement toward the metal andinner-sphere coordination of the phosphate to the active site manganeseion.

Based upon the structures of PGA and 3-phosphonopropionate, we predictthat 2,2-dimethylsulfoacetate, 1,2-ethanediphosphonate, and3-sulfopropionate would bind in a similar outersphere/PEP-likeconformation, resulting in their observed millimolar K_(i) values. Itappears that PEP obtains its higher affinity for the enzyme whencompared to the other PEP analogues, as represented by PGA and3-phosphonopropionoate (89 μM vs 1-2 mM, Table 3), by taking advantageof aromatic interactions with Y235 and/or F333. Both PGA and3-phosphonopropionate lack the C3 methylene group, which appears to makea favorable aromatic interaction with F333. This interaction alsoorients the carboxylate of PEP to favorably interact with Y235. Thestructural data suggest that the loss of the F333 interaction in PGA and3-phosphonopropionate results in a general shift in the position of thebound inhibitor as it coordinates directly to the manganese ion, and asa result, the edge on aromatic-carboxylate interaction with Y235 iseliminated or at least significantly decreased.

While a priori phosphonoformate was predicted to be a PEP analogue, thestructural data demonstrate that it mimics the binding of OAA.Consistent with this observation is its micromolar K_(i) value similarto the K, of oxalate. The structural data show that in phosphonoformatethe juxtaposition of the two carbonyl oxygens of the phosphonate and thecarboxylate allows it to mimic the planar cis sp² central skeleton ofOAA/oxalate, thus making it an effective analogue of OAA that bindsdirectly to the manganese ion. Similar to oxalate, the additionalinteractions of the other phosphono and carboxylate oxygens with R87,S286, and R405 appear to result in the observed micomolar bindingaffinity.

Sulfoacetate is an outlier that utilizes motifs of both OAA and PEPrecognition to achieve a reasonable binding affinity. Thus, whilebinding in a position similar to PEP that results in the edge on Y235carboxylate interaction, the sulfo group does not mimic the outerspherecoordination of the phosphate of PEP or the innersphere coordination ofPGA or phosphonopropionate. Instead, the sulfo group mimics the C1carboxylate of OAA, interacting in a similar fashion with R87 and R405.

Together the kinetic and structural data suggest the following motifsfor substrate/inhibitor recognition (FIGS. 2A-2B): Cis-planar sp²carbonyl moieties facilitate the coordination of the ligand directly tothe active site metal. As suggested by the binding of OAA, oxalate andphosphonoformate, (1) the terminal carbonyl can be a carboxylate inorder to facilitate interaction with S286 (FIGS. 1A-1F); (2) a bridgingoxygen or similar electron rich atom between the C1 carboxylate and C3carbonyl of OAA allows for tighter binding by exploiting electrostaticand hydrogen bonding possibilities with R405; (3) carboxylate orsulfonate at the position corresponding to the C1 carboxylate of OAAexploits further interactions with R87 and R405; and (4) aromatic andhydrogen bonding interactions at a position corresponding to the C1carboxylate of PEP and Y235 and N403 respectively appear to beresponsible for at least an order of magnitude increase in bindingefficiency.

At best, all of the molecules tested that are effective inhibitors(e.g., sulfoacetate, oxalate, and phosphonoformate) or substrates (e.g.,PEP, OAA) appear to take advantage of only two of the possibleinteractions described above in order to achieve the observed micromolarK_(i) or K_(m) values. In theory, it should be possible to obtain quitehigh affinities and specificity if all four interactions are exploited,based upon the observed differences in inhibition by pyruvate andoxalate discussed previously. In addition, the binding of sulfoacetatesuggests that larger molecules based upon the structure of OAA thatcould extend in the active site and form the favorable interactions withY235, F333 and N403 would be potent and selective PEPCK inhibitors.

In conclusion, this study provides the first structure-function analysisof the PEP/OAA binding site of mammalian PEPCK and illustrates themechanism of substrate/inhibitor molecular recognition utilized byPEPCK, which can be exploited in the design of effective and selectiveinhibitors of PEPCK. A summary of the figures is provided below.

FIG. 1A shows the interaction of oxalate at the PEPCK active site. Thebicarboxylate structure is specifically recognized by directcoordination of two of the carboxylate oxygens through directcoordination to the active site manganese ion (2.16 and 2.31 Årespectively) as well as hydrogen bonding of one of the oxygens toserine 286 (S286). The other carboxylate oxygens form discrete hydrogenbonds with R405 and R87 (2.93, 3.06 and 2.78 Å).

FIG. 1B shows the interaction of phosphonoformate at the PEPCK activesite. The C1 carboxylate interacts in an identical fashion with theenzyme as the similar carboxylate in oxalate shown in FIG. 1A. Two ofthe oxygens of the phosphono group are able to make similar interactionswith S286 and R87 however the later interactions occurs through the NEof R87 rather than the terminal amino group as observed in the oxalatecomplex.

FIGS. 1C and 1D show the two binding orientations for phosphoglycolate(PGA) observed at the active site of PEPCK. PGA is observed to bind intwo different orientations. In C) one phosphoryl oxygen is observed tocoordinate directly to the active site manganese ion (2.15 Å) in asimilar fashion to the C1 carboxylate of phosphonoformate and oxalate inpanels A and B. In the conformation shown in D, PGA adopts the sameouter-sphere conformation with the active site manganese ion as seenwith the substrate PEP indirectly interacting with the metal ion throughtwo water molecules coordinated to the metal ion. In both orientationsthe other phosphoryl oxygen atoms make favorable interactions with theguanidinium groups of R87 and R405 however as depicted the specificcontacts difference between the two conformers. In both orientations,the C2 carboxylate extends away from the manganese ion towards the backof the binding pocket making discrete interactions with the backbonenitrogen of residue 237 as well as the backbone nitrogen of R87 and theside chain of asparagine 403(N403) in the case of the conformationobserved in D). In both orientations the side chain of tyrosine 235(Y235) is observed in its rearward conformation and the C2 carboxylatemakes a favorable edge on carboxylate-aromatic interaction.

FIG. 1E shows the interaction of phosphonopropionoate at the PEPCKactive site. Phosphononopropionate interacts in an identicalconformation to that observed in panel C for PGA. The lack of the secondbinding conformation is due to the absence of the bridging oxygen inphosphonopropionate that makes the favorable interaction (3.22 Å) withthe amino group of R405 in panel D. Further the lack of the bridgingoxygen prevents the molecule from adopting an additional conformationthat occurs upon the formation of the PGA-GDP nucleotide complex(Sullivan and Holyoak, 2008) resulting in less dynamic motion in theinhibitor as the lack of the bridging oxygen in phosphonopropionateprecludes this additional conformation in the presence of GDP nucleotide(unpublished data).

FIG. 1F shows the interaction of sulfoacetate at the PEPCK active site.Unlike the other inhibitors, sulfoacetate is observed to occupy only therearward pocket of the PEPCK active site with no interaction with theactive site manganese ion. The C2 carboxylate interacts with the enzymein an identical fashion to the PGA conformation shown in panel D whilethe C1 sulfate group occupies the CO2 binding site of the substrate OAA(Sullivan and Holyoak, 2007) through hydrogen bonding interactions withR87 and a salt bridge with R405.

FIGS. 2A-2B show bound oxalate and sulfoacetate are shown as stickmodels demonstrating the boundaries of the OAA/PEP binding site. Theportions labeled by (2) correlates to a negative charge, and elementsidentified by (3) are negatively charged. The portions labeled with (4)correlates to a positive charge, and elements identified by (5) arepositively charged. The non-overlapping nature of the two inhibitorsdefines the two subsites in the OAA/PEP binding site of the PEPCK activesite. All of the residues that can directly interact with an inhibitormolecule that occupies both of these subsites simultaneously are shownand labeled. Connection of these two individual subsites in a singlemolecular scaffold will lead to potent and selective inhibitors of PEPCKas anti-hyperglycemic agents.

FIG. 3 shows a novel allosteric binding site for regulation of PEPCKactivity, such as PEPCK inhibition. Crystallographic studies ofcytosolic PEPCK illustrate that a novel binding site for the molecule3-mercaptopicolinic acid (MPA) exists in a unique pocket adjacent to theactive site of PEPCK. This site is framed by the active site P-loop andthe loop composed of residues 514-533 (FIG. 3). Kinetic data demonstratethat MPA binds to this site and is competitive with nucleotide bindingwith an apparent affinity of ˜100 μM. Analysis of the crystallographicdata from several complexes of PEPCK with MPA and various substrates andsubstrate analogues illustrates that binding to this allosteric siteresults in a conformational change involving residue F517 and moregenerally an alteration in the position of 514-533 loop. Theseconformational changes distort the nucleotide-binding pocket andpreclude the binding of nucleotide to PEPCK resulting in catalyticinhibition. While the inhibition of PEPCK by MPA has been previouslydescribed, this work is the first to identify the novel allostericbinding site and describe the allosteric mechanism of inhibition by thiscompound. Knowledge of this binding site can now be utilized to guidethe design of molecules with a high affinity for this binding site inthe development of anti-hyperglycemic agents targeting PEPCK in thetreatment of diabetes-associated hyperglycemia.

FIG. 4 shows that CMMP (e.g., Formula C) is an effective PEPCKinhibitor. Due to the partially overlapping binding sites for PEP andOAA prior to lid closure, obtaining both selectivity and potency inactive site inhibitors should be possible because of the uniquesub-sites existing within the PEP/OAA binding site due to the dynamicstructural changes induced by lid opening and closing. This wasoriginally supported by the inhibition of PEPCK by sulfoacetate. In thatwork we demonstrated that sulfoacetate obtains a reasonable level ofinhibition (K_(i)=82 μM) by bridging the two active site sub-sites andexploiting interactions utilized in the binding of both OAA and PEP.Based upon that work, we proposed a novel scaffold to be utilized in thedevelopment of molecules that bridge these two sub-sites with and indoing so would gain potency and, perhaps more importantly with ametabolic enzyme like PEPCK, specificity. This scaffold is Formulas C,E, and/or G and an example is CMMP. Kinetic analysis demonstrates thatCMMP is a competitive inhibitor against PEP in the PEPCK reaction. Itexhibits a reasonable K_(i) (12 μM) and the crystal structure of thePEPCK-CMMP complex illustrates that the molecule binds exactly aspredicted by our previous studies, bridging the OAA and PEP sub-sites(FIG. 4). This molecule thereby gains both potency and specificity forPEPCK. FIG. 5 shows the inhibition data for CMMP against PEPCK, wherethe data illustrates the inhibition of PEPCK by CMMP and demonstratethat it is competitive in its binding with PEP. The information obtainedfrom FIGS. 4 and 5 indicate that derivatives of CMMP, as shown by thescaffolds of Formulas E and G, are likely to be similarly effective asCMMP.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope. All references recitedherein are incorporated herein by specific reference (Sullivan, S. M.and Holyoak, T. Enzymes with lid-gated active sites must operate by aninduced fit mechanism instead of conformational selection. Proc NatlAcad Sci USA 2008; 105:13829-34; Sullivan, S. M. and Holyoak, T.Structures of rat cytosolic PEPCK: Insight into the mechanism ofphosphorylation and decarboxylation of oxaloacetic acid. Biochemistry2007; 46:10078-88).

Tables

TABLE 1 Substrate Analogues of Rat Liver Cytosolic PhosphoenolpyruvateCarboxykinase. % Act- ivity Re- main- # Name Chemical Formula ing^(a)Dicarboxylates  1 Oxalate^(b) CO₂ ⁻CO₂ ⁻  11^(c)  2 Malonate^(b) CO₂⁻CH₂ CO₂ ⁻ 102^(c)  3 Succinate^(b) CO₂ ⁻CH₂CH₂ CO₂ ⁻  91^(c)  4Sulfosuccinate^(d) CO₂ ⁻CH(SO₃ ⁻)CH₂ CO₂ ⁻  55^(c)  5 Maleate CO₂ ⁻CH:CHCO₂ ⁻ (cis)  52^(e)  6 Fumarate CO₂ ⁻CH:CH CO₂ ⁻ (trans)  83^(e)  7Itaconate CO₂ ⁻CH₂C(:CH₂) CO₂ ⁻  91^(c)  8 1,2-Cyclopen-tanedicarboxylate

 91^(c)  9 L-Aspartate^(b,d) CO₂ ⁻CH₂CH(NH₃ ⁺)CO₂ ⁻ 112^(c)Phosphonyl/phosphoryl monocarboxylates 10 Phosphono- PO₃ ²⁻CO²⁻  12^(c)formate^(b) 11 Phosphono- PO₃ ²⁻CH₂CO²⁻ 108^(c) acetate^(b) 123-phosphono- PO₃H⁻CH₂CH₂ CO²⁻  57^(c) propionate 13 Phosphogly- PO₃²⁻OCH₂ CO²⁻  25^(c) colate^(b) 14 2-D-Phospho- PO₃ ²⁻OCH(CH₂OH) CO²⁻110^(c) glycerate^(b,d) 15 2-(Phosphono- PO₃H⁻CH₂C(:CH₂) CO²⁻  92^(c)methyl)acrylate 16 6-phospho- PO₃ ²⁻OCH₂(CHOH)₄ CO²⁻ 117^(c)gluconate^(b) 17 N-Phosphono- PO₃H⁻CH₂NHCH₂ CO²⁻ 104^(c)methylglycine^(b) 18 2-Amino-3-phos- PO₃H⁻ CH₂CH(NH₃ ⁺)(CO²⁻)  94^(c)phonopropionate^(d) 19 2-Amino-4-phos- PO₃H⁻(CH₂)₂CH(NH₃ ⁺)(CO²⁻)104^(c) phonobutyrate^(b) 20 2-Amino-5-phos- PO₃H⁻(CH₂)₃CH(NH₃ ⁺) CO²⁻ 98^(f) phonovalerate^(d) 21 Serine phosphate^(d) PO₃ ²⁻OCH₂CH(NH₃ ⁺)CO²⁻ 108^(f) 22 Threonine PO₃ ²⁻OCH(CH₃)CH(NH₃ ⁺) CO²⁻  97^(c)phosphate^(d) Sulfonyl/sulfinyl monocarboxylates 23 Sulfoacetate SO₃⁻CH₂ CO²⁻  18^(f) 24 2,2-Dimethyl- SO₃ ⁻C(CH₃)₂ CO²⁻  65^(c)sulfoacetate 25 3-Sulfopropionate SO₃ ⁻CH₂CH₂ CO²⁻  69^(c) 26 Cysteicacid^(d) SO₃ ⁻CH₂CH(NH₃ ⁺) CO²⁻  76^(c) 27 Cysteine SO₃ ⁻CH₂CH(NH₃ ⁺)CO²⁻ 105^(c) sulfinic acid^(d) Diphosphoryls 28 Pyrophosphate^(b) PO₃H⁻OPO₃H⁻  22^(f) 29 Methanedi- PO₃H⁻CH₂ PO₃H⁻  4^(f) phosphonate 301,2-Ethanedi- PO₃H⁻CH₂CH₂ PO₃H⁻  96^(e) phosphonate Disulfonates 31Methane- SO₃ ⁻CH₂ SO₃ ⁻  4^(f) disulfonate 32 1,2-Ethane- SO₃ ⁻CH₂CH₂SO₃ ⁻  42^(e) disulfonate Epoxy/aromatic compounds 33 Phosphomycin

108^(c) 34 Phenylphosphate C₆H₅OPO₃ ²⁻  96^(c) 35 p-Nitrophenyl-NO₂C₆H₄OPO₃ ²⁻ 103^(c) phosphate ^(a)In this preliminary screen, theinhibition by each compound was tested twice using two differentpreparations of PEPCK. The remaining percent activity listed belowrepresents the average of the two values. ^(b)These compounds have beenpreviously evaluated as either reversible inhibitors or alternativesubstrates for rat liver cytosolic PEPCK and other PEP-utilizing enzymes(see text for references). ^(c)Activity was determined at analogue andPEP concentrations of 3 mM and 40 μM, respectively. ^(d)For theanalogues containing a chiral center, the chiral carbon is italicized.^(e)Activity was determined at analogue and PEP concentrations of 6 mMand 40 μM, respectively. ^(f)Activity was determined at analogue and PEPconcentrations of 1.5 mM and 40 μM, resepctively.

TABLE 2 Data and Model Statistics for the PEPCK-Mn²⁺-oxalate,PEPCK-Mn²⁺- phophonoformate, PEPCK-Mn²⁺-PGA,PEPCK-Mn²⁺-phosphonopropionate and PEPCK-Mn²⁺-sulfoacetatecomplexes^(a). PEPCK-Mn²⁺- PEPCK-Mn²⁺- PEPCK-Mn²⁺- PEPCK-Mn²⁺-PEPCK-Mn²⁺- oxalate phosphonoformate PGA phosphonopropionatesulfoacetate wavelength (Å) 1.54 1.54 1.54 1.54 1.54 space group P2₁ P2₁P2₁ P2₁ P2₁ unit cell a = 64.0 Å a = 62.3 Å a = 60.7 Å a = 45.3 Å a =45.2 Å b = 118.9 Å b = 119.5 Å b = 119.7 Å b = 119.4 Å b = 119.0 Å c =86.5 Å c = 86.9 Å c = 90.9 Å c = 60.8 Å c = 60.9 Å α = γ = 90.0° α = γ =90° α = γ = 90° α = γ = 90° α = γ = 90° β = 107.0° β = 107.1° β = 108.9°β = 108.7° β = 108.8° resolution limit (Å) 30.8-1.9 30.1-2.00 29.9-1.9534.0-1.9 29.1-1.8 no. of unique 91629 76020 81978 44417 50438reflections Completeness^(b) (%; 99.1 (93.6) 98.2 (91.1) 96.4 (93.0)97.1 (81.3) 94.4 (64.9) all data) redundancy^(b) 5.5 (4.7) 5.7 (4.5) 6.8(6.3) 7.0 (5.5) 8.5 (3.7) I/σ_((I)) ^(b) 11.9 (2.1)  9.7 (1.9) 16.1(2.6)  20.2 (2.4)  19.5 (1.7)  R_(merge) ^(b,c) 0.09 (0.52) 0.10 (0.52)0.07 (0.53) 0.07 (0.53) 0.08 (0.45) no. of ASU 2 2 2 1 1 moleculessolvent content (%) 40.1 39.1 38.4 39.9 39.9 R_(free) ^(b,d) (%) 25.6(35.2) 24.9 (34.5) 22.2 (33.3) 23.3 (36.2) 24.9 (36.8) R_(work) ^(b,e)(%) 20.7 (28.3) 19.8 (27.2) 18.5 (25.4) 18.9 (30.4) 20.5 (34.7) averageB values^(f) 10.9 18.1 18.4 14.1 12.1 protein water 25.1 31.1 26.7 28.422.9 inhibitor Oxalate Phosphonoformate PGA PhosphonopropionateSulfoacetate Mol A Mol A Mol A 19.4 18.5 18.0 16.4 PGA₁ = 15.7, Mol BMol B oc = 0.5 18.6 25.0 PGA₂ = 22.1, oc = 0.5 Mol B PGA₁ = 14.4, oc =0.5 PGA₂ = 19.7, oc = 0.5 estimated 0.125 0.143 0.115 0.122 0.118coordinate error based on maximum likelihood (Å) bond angle rmsd 1.1191.117 1.125 1.112 1.237 (deg) Ramachandran 90.5 90.8 91.2 91.0 91.4statistics (most 8.7 8.6 8.2 8.2 7.8 favored, 0.6 0.3 0.6 0.8 0.8additionally 0.2 0.3 0 0 0 allowed, generously allowed, disallowed) (%)^(a)Mol A; molecule A of the crystallographic dimer Mol B; molecule B ofthe crystallographic dimer oc; ligand occupancy PGA₁ and PGA₂ correspondto the two alternate conformations of PGA present in each molecule ofthe PEPCK-Mn²⁺-PGA crystallographic dimer. ^(b)Values in parenthesesrepresent statistics for data in the highest-resolution shells. Thehighest-resolution shell comprises data in the range of 1.97-1.90,2.07-2.00, 2.02-1.95, 1.97-1.90, and 1.85-1.80 Å for thePEPCK-Mn²⁺-oxalate, PEPCK-Mn²⁺-phosphonoformate, PEPCK-Mn²⁺-PGA,PEPCK-Mn²⁺-phosphonopropionate, and PEPCK-Mn²⁺-sulfoacetate data sets,respectively. ^(c)R_(merge) = Σ|I_(obs) − I_(avg)|/ΣI_(obs) ^(d)SeeBrunger (36) for a description of R_(free). ^(e)R_(work) = Σ||F_(obs)| −|F_(calc)||/Σ|F_(obs)| ^(f)B values indicated are residual B valuesafter TLS refinement.

TABLE 3 Inhibition Constant for PEP and OAA Analogues. Analogue K_(i)Pattern of Inhibition Oxalate (1) 89 ± 4 μM^(a) Competitive Succinate(3) >8.0 mM^(b) n.d.^(c) Maleate (5) 2.0 mM^(b) n.d.^(c)Phosphonoformate (10) 230 ± 14 μM^(a) Competitive Phosphoglycolate (13)1.0 ± 0.04 mM^(a) Competitive 3-Phosphonopropionate (12) 1.9 ± 0.4mM^(a) Competitive 1,2-Ethanediphosphonate (30) 5.1 ± 0.5 mM^(a)Competitive Sulfoacetate (23) 82.5 ± 5 μM^(a) Competitive2,2-Dimethylsulfoacetate (24) 2.1 ± 0.2 mM^(a) Competitive3-Sulfopropionate (25) 3.4 ± 0.3 mM^(a) Competitive Sulfosuccinate (4)3.3 mM^(b) n.d.^(c) 1,2-Ethanedisulfonate (32) 3.0 mM^(b) n.d.^(c)Pyrophosphate (28) 34 ± 5 μM^(a,d) Noncompetitive^(f) 64 ± 7 μM^(e) 172± 29 μM Competitive^(g) Methanediphosphonate (29) 32 ± 2 μM^(a,d)Noncompetitive 90 ± 5 μM^(e) Methanedisulfonate (31) 27 ± 3 μM^(a,d)Noncompetitive 168 ± 25 μM^(e) ^(a)The K_(i) values were obtained usingthe Cleland kinetics program for competitive or noncompetitiveinhibitors. ^(b)The K_(i) values were obtained from Dixon plots usingtwo PEP concentrations. ^(c)Not determined ^(d)Slope effect^(e)Intercept effect ^(f)PEP was the varied substrate. ^(g)IDP was thevaried substrate.

1. A compound for inhibiting phosphoenolpyruvate carboxykinase (PEPCK)in a subject, the compound comprising: a molecule configured forinteracting with a biding site of PEPCK so as to inhibit PEPCK, saidmolecule being characterized by having a size capable of fitting intoand interacting with the PEPCK binding site and at least one of thefollowing: (a) a first terminal substituent having co-planar atomsacting as metal ligands to the active site metal ion of PEPCK; (b) atleast one of an atom or substituent at positions 2 or 3 from the firstterminal substituent includes a neutral carbon center or include anoxygen, sulfur, selenium, or other atom with similar physiochemicalproperties, (c) at least one of an atom or substituent at positions 2 or3 from the first terminal substituent is devoid of an electropositiveatom or substituents; or (d) a second terminal substituent opposite ofthe first terminal substituent, said second terminal substituent havingan atom that is a hydrogen boding acceptor and/or is negatively charged.2. A compound as in claim 1, wherein the PEPCK inhibitor includes atleast two of (a), (b), (c), or (d).
 3. A compound as in claim 2, whereinthe PEPCK inhibitor includes at least three of (a), (b), (c), or (d). 4.A compound as in claim 3, wherein the PEPCK inhibitor includes (a), (b),(c), and (d).
 5. A compound as in claim 1, wherein at least one of theco-planar atoms of the first terminal substituent interacts with S286.6. A compound as in claim 1, wherein the compound interacts with atleast one of R87 or R405.
 7. A compound as in claim 1, wherein the PEPCKinhibitor includes features one of Formula A, B, C, or D, or saltthereof, acid thereof, derivative thereof or combinations thereof so asto interact with M2+ active metal site of PEPCK through interactions (a)and (b):


8. A compound as in claim 7, wherein the PEPCK inhibitor has a structureof Formula C or derivative or salt thereof.
 9. A compound as in claim 1,wherein the PEPCK inhibitor has a structure of Formula E or derivativeor salt thereof:

wherein: R1 is a hydrogen, halogen, Cl, F, CH₃, CH₃CH₂, or higher orlower substituted or unsubstituted straight chain or branched aliphatic(e.g., C1-C10); R2 is hydrogen, CH₃CH₂, or C1-C10 substituted orunsubstituted straight chain or branched aliphatic; R3 is a carboxylicacid, amide, Formula F, sulphate, phosphate, or other hydrogen bonddonor or has a positive charge; X can be C, O, or N, such that X is ahydrogen bond donor; and n can be 0, 1, 2, or
 3. 10. A compound as inclaim 9, wherein at least one of R1 or R2 is electronegative and notelectropositive.
 11. A compound as in claim 9, wherein the PEPCKinhibitor has a structure of Formula G or derivative or salt thereof:


12. A compound as in claim 1, wherein the PEPCK inhibitor is devoid ofbeing characterized by at least one of the following: a methyl ormethylene center which is incapable of interacting with R405; a sizeincapable of fitting within the binding pocket framed by R87, K244,G237, F333, R405, N403 and/or Y235; a steric conflict with F333; or apositively charged functional group incompatible with a positivelycharged active site of PEPCK.
 13. A composition for inhibitingphosphoenolpyruvate carboxykinase (PEPCK) in a subject, the compositioncomprising: a pharmaceutically acceptable carrier; and a therapeuticallyeffective amount of the PEPCK inhibitor of claim
 1. 14. A compositionfor inhibiting phosphoenolpyruvate carboxykinase (PEPCK) in a subject,the composition comprising: a pharmaceutically acceptable carrier; and atherapeutically effective amount of the PEPCK inhibitor of claim
 8. 15.A composition for inhibiting phosphoenolpyruvate carboxykinase (PEPCK)in a subject, the composition comprising: a pharmaceutically acceptablecarrier; and a therapeutically effective amount of the PEPCK inhibitorof claim
 9. 16. A method for inhibiting phosphoenolpyruvatecarboxykinase (PEPCK) in a subject, the method comprising: administeringto the subject a composition having a PEPCK inhibitor characterized byhaving a size capable of fitting into and interacting with the PEPCKbinding site and at least one of the following: (a) a first terminalsubstituent having co-planar atoms acting as metal ligands to the activesite metal ion PEPCK; (b) at least one of an atom or substituent atpositions 2 or 3 from the first terminal substituent includes a neutralcarbon center or include an oxygen, sulfur, selenium, or other atom withsimilar physiochemical properties, (c) at least one of an atom orsubstituent at positions 2 or 3 from the first terminal substituent isdevoid of an electropositive atom or substituents; or (d) a secondterminal substituent opposite of the first terminal substituent, saidsecond terminal substituent having an atom that is a hydrogen bodingacceptor and/or is negatively charged.
 17. A method as in claim 16,where the PEPCK inhibitor is administered in a therapeutically effectiveamount for treating, inhibiting, and/or hyperglycemia in the subject.18. A method as in claim 17, wherein the subject is a diabetic patient.19. A method as in claim 16, wherein the PEPCK inhibitor has a structureof Formula E or derivative or salt thereof:

wherein: R1 is a hydrogen, halogen, Cl, F, CH₃, CH₃CH₂, or higher orlower substituted or unsubstituted straight chain or branched aliphatic(e.g., C1-C10); R2 is hydrogen, CH₃CH₂, or C1-C10 substituted orunsubstituted straight chain or branched aliphatic; R3 is a carboxylicacid, amide, Formula F, sulphate, phosphate, or other hydrogen bonddonor or has a positive charge; X can be C, O, or N, such that X is ahydrogen bond donor; and n can be 0, 1, 2, or
 3. 20. A method as inclaim 19, wherein the PEPCK inhibitor has a structure of Formula G orderivative or salt thereof:


21. A method as in claim 19, wherein the PEPCK inhibitor has a structureof Formula C: